CN105228628A - Synthesis and the compositions of base is connected with the aminoacid for making the compound of cancer target imaging put together - Google Patents

Abstract

The present invention relates to the compound as near infrared fluorescent probe, wherein this compound comprises: i) in conjunction with the pteroyl-part of target receptor protein; Ii) dye molecule; And iii) connection molecule containing aminoacid or derivatives thereof.Invention further describes preparation and use the method and composition of described compound, mix the method for described compound and mix the test kit of described compound.

Description

Synthesis and the compositions of base is connected with the aminoacid for making the compound of cancer target imaging put together

Related application

Present patent application relates to and requires the U.S. Provisional Patent Application serial number US61/791 that on March 15th, 2013 submits to, the benefit of priority of 921, by document content intact to introduce the application for referencial use.

Technical field

The application belongs to diagnostic field.The application provides synthesis and uses aminoacid to be connected the method for base, and described aminoacid connects base and for making the compound of cancer target imaging put together.Put together aminoacid and connect specificity and the detection property that base adds described compound.Focus on the method and composition applied in diagnosing image.

Background technology

Operation removing malignancy disease constitutes one of the most frequently used and effective treatment means for cancer Primary treatment.The excision of all malignant tumor lesions detected causes cannot not detecting that this disease recurs in all cancer patients' about 50% ¹, and the sickness rate that the patient of cancer return is observed in life expectancy or minimizing may be extended.Two people are not in surprise, are the more investigations accepted at present for realizing the operation method of more quantitative cytoreduction.

The excision of all malignant tumor lesions detected causes cannot not detecting that this disease recurs in all cancer patients' about 50%, and may extend the sickness rate that the patient of cancer return is observed in life expectancy or minimizing.Due to the importance of the infringement that removes the malignant tumour completely, so it is beneficial that guarantee accurately and fully to identify malignant tumor lesions.In operation process, identify that malignant tumor lesions is undertaken by three kinds of methods at present.First, can based on abnormal color, quality and/or form by the many tumor mass of vision-based detection and root nodule.Therefore, tumor mass can demonstrate mottled color, occurs the unsymmetry or outstanding from the profile of healthy organ with irregular obstacle body.Malignant tumor block can also because of flexibility, elasticity or flintiness by sense of touch and adjacent health tissues identification.Finally, can use fluorescent dye in operation process, locate several cancer focus, described fluorescent dye flows into the lymph node of drain passively from primary tumor.In the method in face in this post, visual identification fluorescence (labelling) lymph node can be passed through, excision and check whether cancerous cell is transferred to these lymph nodes.

Although recognize the importance of removing tumor with some authenticate technologies in the utilizability making tumor mass develop, many malignant tumor root nodules have still escaped from detection, cause palindromia and usually cause death.Therefore, demand is existed to the tumor qualification improved.This motivation has caused introducing two kinds of new methods for making malignancy disease develop in performing the operation.In first method, the fluorescent dye of quencher is injected the animal with tumor by whole body, and be used in by tumour-specific enzyme, pH change or oxidation-reduction potential change release quencher moieties the fluorescence optionally activated in malignant tumor block.In the second approach, fluorescent dye is puted together in tumour-specific targeting part, described tumour-specific targeting part causes the dyestuff combined to be accumulated in the cancer of the receptor of part described in overexpression.Example for the cancer target part of the object after this comprises folic acid, its display is to the specificity of folacin receptor (FR) positive cancer of ovary, kidney, lung, endometrium, mammary gland and colon and DUPA, the fluorescent dye of connection optionally can be delivered to the cell of expressing prostate specific membrane antigen (PSMA) by it, i.e. the neovasculature of carcinoma of prostate and other solid tumor.Valuably, in human ovarian cancer patient, test a kind of fluorescent dye (folic acid-fluorescein or EC17) of folate-targeted in the recent period.In this study, eliminate malignant tumor lesions ~ more than the 5X exceeding and do not use it by means of cancer target fluorescent dye, and the infringement of the fluorescence of all excisions is all proved to be malignant tumor by pharmacological method.

Conventional fluorescent technique is used in the probe in visible light (~ 400-600nm), it to instruct for surgical operation and non-optimal for image in operation process, because it is relevant to non-specific background's light of the relative high levels caused because of collagen protein in organizing.Therefore, the signal to noise ratio from these Conventional compounds is low.In addition, by biological chromophore, particularly hemoglobin absorption visible ray, the depth of penetration is limited to several millimeter.Therefore, bury in tissue the tumor being deeper than several millimeters may still cannot detect.In addition, the ionization equilibrium (pKa=6.4) of fluorescein causes absorbing at the pH-dependency of 5-9 scope and launching.Therefore, based on the fluorescence of the dyestuff of fluorescein under low pH (lower than pH5) by quencher.

Such as, EC17 dyestuff is more being widely used in optical imagery to characterize with the potential application in the illing tissue measured in clinical setting because the dyestuff (fluorescein) that is connected is obstructed at the major defect of visible-range emitting fluorescence.This makes EC17 and related dye be difficult to be applied to in-vivo tissue, typically sends autofluorescence consumingly and light is difficult to penetrate tissue at visible-range because organize.In addition, EC17 (folic acid-ethylenediamine-Fluorescein isothiocyanate) constitutes thiourea connection base.Well-known thiourea compound has the low shelf life because of the unstability of thiourea key.Therefore, compound such as EC17 for being applied to for optical imagery and non-optimal, this correlation down decomposed owing to this unstability and thiourea bridge.

The combination of the water in visible spectrum (<600nm) in hemoglobin and IR scope (>900nm) and the light absorption of lipid provides the optical imagery window of about 650-900nm, and the absorptance wherein organized is minimum.The dyestuff that research and development may be used for near-infrared (NIR) scope is may be used for, because the inducing self-body fluorescence and effectively penetrate tissue hardly of the light near infrared region in the alternative selection be applicable to of the radiative dyestuff of visible range.The background of scattered light that closely another helpfulness of-IR fluorescent technique is since excitaton source obviously reduces, because scattering strength is directly proportional to the quadruplicate inverse of wavelength.Low background fluorescence is that high-sensitivity detection is necessary.In addition, optical clear observation port line (opticallytransparentwindow) in biological tissue in nearly-IR district (650nm to 900nm) makes NIR fluorescence become the valuable technology detecting application for in-vivo imaging and subcellular fraction, and described in-vivo imaging and subcellular fraction detect application needs light by biotic component transmission.

Although light for the application in the NIR scope of darker imaging of tissue preferentially for the light in visible spectrum, but there is a large amount of challenge and defect in the NIR image-forming dye used in the art, such as photobleaching sensitivity, poor chemical stability, fall into and the Absorption and emission spectra of many physiological molecules same range (causing high background signal and autofluorescence).In addition, most of NIR dyestuff is unstable in building-up process, is especially connected base with amine and puts together, produce multiple unwanted by-product.Therefore, the NIR preparation adopting part-targeting may be expensive for clinical.So, use the fluorescence signal of the formation method of NIR fluorescent probe in Form (apart from surperficial >5mm) imaging, mammalian tissues quantitative or invalid in increase preclinical phase to the production cost of clinical conversion time at present.

The method of two kinds of richness fluorescence likely guided surgeries is at present in clinical anxiety research.In one approach, activable NIR fluorescent probe sends minimum fluorescence because of it close to the quencher connected in stable state, becomes obvious fluorescence when it discharges quencher in malignant tumor tissue.One of the most frequently used releasing mechanism is involved between dyestuff and quencher and is incorporated to state sequence, and it can specifically by protease (i.e. cathepsin, Caspase and matrix metalloproteinase) cracking that tumor is rich in.The major advantage of this strategy is to there is not fluorescence in the tissue lacking activating enzymes, makes tissue retain non-fluorescence, unless they unexpectedly express described lyases along excretion pathway (such as kidney, bladder, liver).The NIR dyestuff of this tumor-activated can also generate a large amount of fluorescence in tumor mass, if be rich in described lyases in malignant tumor lesions and release dyestuff be retained in tumor.The major defect of this method is owing to the tumour-specific difference (its major part is also expressed in the health tissues carrying out natural reconstruction or experience inflammation) of many relevant hydrolytic enzyme.In addition, the abundance of the protease expected is variable in tumor mass, causes fluorescence in some malignant tumor lesions slow or without activation, and fluorescence occurs fast in other infringement.

Therefore, still needing can specifically for targeting illing tissue and in vivo for the stability in the application of imaging of tissue with increase and the dye substance measured.

Summary of the invention

The application is provided for synthesizing amino acid and connects the method for base, described aminoacid connect base with for making the compound of tumor and lymph gland targeted imaging put together.In some embodiments, the application relates to the compound or its salt derivant comprising folic acid or pteroyl-part, connection base and fluorescent dye.In some embodiments, described connection base can be aminoacid, its isomer, derivant or racemic mixture.In other side, described fluorescent dye is selected from LS288, IR800, SP054, S0121, KODAK, S2076 and S0456.

In some respects, the application provides the method that aminoacid connection base and fluorescent dye are puted together, and wherein said aminoacid can be tyrosine, serine, threonine, lysine, arginine, agedoite, glutamine, cysteine, selenocysteine, its isomer and derivant.In some embodiments, described aminoacid, its isomer or derivant comprise-OH ,-NH ₂or-SH functional group, it is to make fluorophor and aminoacid, its isomer or derivant put together when slightly molar excess adds in fluorescent dye.In other embodiments, described aminoacid, its isomer or derivant comprise-OH functional group, and it is measured with increase compound when synthesizing and the fluorescent dye of detection property generates ehter bond.In some embodiments, the application relates to aminoacid and connects puting together of base and fluorescent dye, wherein said aminoacid, its isomer or derivant comprise-SH ,-SeH ,-PoH Huo – TeH functional group, its synthesize time and fluorescent dye generate C-S, C-Se, C-Po or C-Te key.In some respects, the application relates to aminoacid and connects puting together of base and fluorescent dye, and described fluorescent dye has the about 500nm extremely absorption maximum of about 900nm and emission spectra.In other side, described aminoacid connects base and has the fluorescent dye of about 600nm to about 800nm absorption maximum and emission spectra and put together.

In further embodiment, the application is provided for the method that aminoacid connection base and folate ligand are puted together, it is tyrosine, serine, threonine, lysine, arginine, agedoite, glutamine, cysteine, selenocysteine, its isomer or derivant that wherein said aminoacid connects base, and is puted together by two peptide bonds with folic acid.In in other, the application provides the conjugation methods connecting base and folate ligand, wherein said connection base is tyrosine, serine, threonine, lysine, arginine, agedoite, glutamine, cysteine, selenocysteine, its isomer or derivant, and puted together by height-oligopeptide key with folic acid.In other embodiments; the application relates to pteroyl-part and is connected the method that base puts together with aminoacid, and wherein said connection base is tyrosine, serine, threonine, lysine, arginine, agedoite, glutamine, cysteine, selenocysteine, its isomer or derivant.In some respects, connect the carboxylic acid of base and be combined with the α carbon of arbitrary amino acid, increase the specificity of compound for receptor targeted thus.In some embodiments, described aminoacid connects base and gives compound specificity, and wherein the binding affinity of viewed compound to receptor targeted refers to folacin receptor.

In other, described compound has high selectivity for the tumor cell of targeted expression target receptor.

In other embodiments, the application's compound of relating to called after Pte-Tyr-S0456 (OTL-0038) at the directed surgery of image, tumor imaging, lymph node imaging, inflammatory diseases, atherosclerosis, infectious disease, legal medical expert's application, minerals utilization, ophthalmology, gel-colored, DNA sequencing, neurally to dye or purposes in plastic surgery.In other side, Pte-Tyr-S0456 derivant can be Pte-D-Tyr-S0456, Pte-height Tyr-S0456, Pte-β-Gao-Tyr-S0456, Pte-(NMe)-Tyr-S0456, Pte-Tyr (OMe)-S0456, Pte-Tyr (OBn)-S0456, Pte-NHNH-Tyr-OAc-S0456, its salt or derivant.

In other side, the application provides the method for synthesis compound, and wherein protecting group is for avoiding the less desirable reactivity that may generate the group of unwanted compound with non-amino.The method that the application provides produces the finalization compound of the yield had more than 98% purity.

In some respects, the application relates to the compound for making cancer target imaging, and wherein this compound may be used for research, diagnosis or therapeutic purposes.In other embodiments, the application's providing package contains the compositions of imaging compounds and pharmaceutically acceptable carrier, excipient, diluent or salt.

In other side, the application relates to the compound having and be selected from following formula:

Wherein W, X, Y or Z are H, Na or NH ₄ ⁺,

Wherein W, X, Y or Z are H, Na or NH ₄ ⁺,

Wherein W, X, Y or Z are H, Na or NH ₄ ⁺,

Wherein W, X, Y or Z are H, Na or NH ₄ ⁺,

Wherein W, X, Y or Z are H, Na or NH ₄ ⁺,

Wherein tyrosine is β height tyrosine,

Wherein W, X, Y or Z are H, Na or NH ₄ ⁺,

And pharmaceutically acceptable salt.

Accompanying drawing explanation

Fig. 1 take folacin receptor as the first generation folic acid-NIR dye conjugate of target.

Fig. 2 first generation folic acid-NIR conjugate in conjunction with isothermal line.The binding curve of the KB cell of folic acid-NIR dye conjugate and expression folacin receptor.The conjugate of targeting is DyLight680 (triangle), AlexaFluor750 (rhombus) and IR800CW (circle).

There is after Fig. 3: intravenous injection first generation folic acid-NIR dye conjugate the fluoroscopic image of the mice (experimental model) of metastatic disease for 4 hours.Complete (A-D) and perform the operation (a-d) that open covers with the fluorescence of the mice of tumor and white light image.To have express FR the nude mouse of transitivity L1210A tumor by intravenous injection 10nmol folic acid-DyLight680 (A/a) or folic acid-DyLight750 (B/b) and imaging after 4 hours.

Fig. 4: the H & E of the tissue of tumor debulking surgery removed during surgery analyzes continuously.After A-D: tail vein injection 10nmol folic acid-IR800CW, 4 hours fluorescence with the mice of L1210A metastatic tumo(u)r and white light image cover.A: whole body images.B: the thoracic cavity of opening.C: after removing primary tumor.D: after removing all secondary lymphoid nodule.The H & E of a-d:a dyes: normal healthy controls lung; B: primary tumor; C: secondary lymphoid nodule.D: residual tissue.

Fig. 5 take folacin receptor as the second filial generation folic acid-NIR dye conjugate of target.

Fig. 6 be the cancerous cell of folic acid-connection base and second filial generation folic acid-NIR dye conjugate and cultivation (use the competitiveness research of radiolabeled folic acid in conjunction with isothermal line.This test provides binding affinity to folacin receptor and specificity simultaneously).The binding curve of the KB cell of A: folic acid-EDA-and B: folic acid-Lys-NIR dye conjugate and expression folacin receptor.

The fluoroscopic image of Fig. 7 second filial generation folic acid-NIR conjugate and in vitro tissue bio distribution.There is after A: intravenous injection 10nmol folic acid-NIR conjugate the fluoroscopic image of the nude mice of KB tumor xenogeneic graft for 2 hours.A () makes the mice group imaging separately of using folic acid-EDA-NIR conjugate; B enemy that () uses the mice of folic acid-EDA-NIR conjugate compares; (c) the mice group imaging separately of using folic acid-Lys-NIR conjugate is made.B: the in vitro tissue bio distribution using the animal of folic acid-NIR conjugate.

Fig. 8 take folacin receptor as the third generation NIR dye conjugate of target.

Fig. 9 Pte-L-Tyr-S0456NIR dye conjugate show structure.There is the chemical constitution of the pteroyl--Tyr-S0456 (OTL-0038) of 4 useful functional groups.A=is as the pteroic acid of target molecule; B=improves α-carboxylic acid to the binding affinity of folacin receptor from tyrosine for tumour-specific; C=is from the phenol moieties of enhancing (highlighting) fluorescence intensity of tyrosine; Nearly-IR the fluorescent probe of d=.Therefore, tyrosine as part, connect base with the part of Jin – IR dyestuff works.In other words, tyrosine improves part (pteroic acid) binding affinity and specific connection base.It also strengthens the brightness of NIR dyestuff.

Figure 10 monitors the reaction process of (A) Pte-Tyr-S0456 (OTL-0038) and (B) folic acid-EDA-IR800CW by LC/MS.Pte-Tyr-S0456 obtains the 99% pure product with the expectation more than 98% yield, and folic acid-EDA-IR800CW obtains the multiple by-product of product with 30-40% expectation.

Figure 11 injects whole body fluorescent image and the in vitro tissue bio distribution of the mice of 10nmolPte-Tyr-S0456.(1) intravenous injection 10nmol take folacin receptor as the fluoroscopic image (fluorescence and white light image cover) 2 hours after the-NIR conjugate of target with the nude mice of KB tumor xenogeneic graft.(2) the in vitro tissue bio distribution of conjugate after the mouse tissue of imaging is in advance gathered.

Figure 12 to nude mice use Pte-Tyr-S0456 and second filial generation folic acid-NIR conjugate (10nmol) afterwards 2h Pte-L-Try-S0456 (OTL-0038) and second filial generation folic acid-NIR conjugate head to head compare (A) whole body fluorescent image; (B) in vitro tissue bio distribution; (C) tumor and kidney image.(section) tumor display target of excision is to preparation uniform pickup in tumor.

Figure 13 have the mice of folate receptor-positive tumor xenogeneic graft used 10nmol often plant conjugate after Pte-Tyr-S0456 to accumulate with the tumor of other pteroyl--NIR dye conjugate and tumour-specific compares.

Figure 14 describes the structure being connected 4 kinds of compounds that base puts together with aminoacid and comprising Pte-Lys-S0456, pteroyl--Cys-S0456, Pte-Ser-S0456 and Pte-4-amino-L-Pro-S0456.

Figure 15 describes OTL-0038, OTL-0039 (the D-isomer of OTL-0038) and folic acid to the RA of folacin receptor.Figure 15 A describes the schematic diagram of often kind of compound to the binding curve of folacin receptor, and Figure 15 B is all binding affinities of 3 kinds of compounds of example and the table of RA.

Figure 16 A describes the whole body fluorescent image with the nude mice of KB tumor xenogeneic graft of injection OTL-0039.To injecting the OTL-0039 of 10nmol in phosphate-buffered saline (100 μ L) in mouse vein.After 2.5 hours, pass through CO ₂suffocate to euthanizing animals.Then the CaliperIVISLuminaIIImagingStation with LivingImage4.0 software is used to carry out whole body imaging experiment.

Figure 16 B example after injection compound 2.5 hours, tissue biological's distribution of the mice of the OTL-0039 in injection Fig. 5 A.After whole body imaging, cut animal and use IVIS imager as above active to tissue (heart, lung, liver,spleen,kidney, stomach, small intestinal, large intestine, muscle, skin and the tumor) analysis of fluorescence selected.

Figure 17 describes the table of the tumor absorption of summarizing OTL-0038.Analyze in the mice of the OTL-0038 of the 0.3-90nmol of injection incremental change and organize bio distribution.Within 2.5 hours, check chorologic data analysis after injection.

Tissue biological's distribution of the mice of the OTL-0038 of Figure 18 exemplary injection incremental change.Used the compound concentration range of 0.3-90nmol to mice by intravenous.Within 2.5 hours, check chorologic data analysis after injection.

The whole body fluorescent image with the nude mice of KB tumor xenogeneic graft of Figure 19 exemplary injection 1nmolOTL-0038.It shows us needs the OTL-0038 of extremely low concentration to make tumor imaging, this is because it is to the more high brightness of the high-affinity of FR and dyestuff.After 2.5 hours, pass through CO ₂suffocate to euthanizing animals.Then the CaliperIVISLuminaIIImagingStation with LivingImage4.0 software is used to carry out whole body imaging experiment.

Figure 20 A describes that to have folacin receptor be the whole body fluorescent image of the mice of tumor xenogeneic graft feminine gender.Within 2.5 hours after using 10nmolOTL-0038, carry out whole body imaging.

The kidney that Figure 20 B uses folacin receptor-negative tumours xenograft and folacin receptor-positive kidney to come example invasive tumor and OTL-0038 absorbs.Inject and carry out data analysis in latter 2.5 hours.

Figure 21 describes and is used for the three-step reaction route that solution is combined to imaging compounds.

Figure 22 describe be used for solid phase synthesis imaging compounds two-step reaction route.

Figure 23 A presents the whole body fluorescent image (injecting latter 2 hours) of the mice of injection 10nmolPte-Tyrosine Analogues-S-456.

Figure 23 B presents tissue biological's distribution (injecting latter 2 hours) of Pte-Tyrosine Analogues-S0456.

Figure 24 shows the whole body fluorescent image (injecting latter 2 hours) of mice of injection 10nmolOTL-0038 (Pte-Tyr-S0456), OTL-0053 (pteroyl--Lys-S0456) and OTL-0054 (pteroyl--Cys-S0456).Excite: 745nm.Launch: 830nm.

Figure 25 shows tissue biological's distribution (injecting latter 2 hours) of OTL-0038 (Pte-Tyr-S0456), OTL-0053 (pteroyl--Lys-S0456) and OTL-0054 (pteroyl--Cys-S0456).Excite: 745nm.Launch: 830nm.

Figure 26 describes whole body and the half body fluoroscopic image (injecting latter 2 hours) of the mice of injection 10nmolOTL-0051 (pteroyl--Tyr-IRD28) and OTL-0052 (pteroyl--Tyr-Kodak).

Figure 27 describes tissue biological's distribution (injecting latter 2 hours) of OTL-0051 (pteroyl--Tyr-IRD28) and OTL-0052 (pteroyl--Tyr-Kodak).

Detailed description of the invention

Operation is one of the best therapy for all solid tumors, such as carcinoma of prostate, ovarian cancer, pulmonary carcinoma, breast carcinoma, colon cancer and cancer of pancreas.In the U.S., although operation is 50% to have in the patient of solid tumor effectively, the effective percentage of independent chemotherapy and radiation in whole cancer is lower than 5%.Have every year more than 700 in the U.S., 000 patient carries out cancer operation, and the patient with operation of 40% had local disease's recurrence in 5 years.Although 10 years inherent oncology aspects achieve major progress in the past, still there is significantly barrier cancer in the art needs to overcome.Such as, be still difficult to excise non-flanged primary tumor completely, remove the lymph node and qualification satellite disease (satellitedisease) of hiding metastatic carcinoma cell.The improvement realizing these three kinds of situations not only improves disease clearance rate, and instructs the decision about postoperative chemotherapy and radiation.Although verified non-targeted fluorescent dye is accumulated in solid tumor passively, the tumor obtained is educated background and may be difficult to determine than usually poor and border between malignant tumor and health tissues.Although the fluorescent dye of part targeting (such as EC17: folic acid-EDA-FITC) is for making imaging of tissue, but those dyestuffs are invalid, because they can not penetrate deep tissue and only identify thus in non-tissue sample compared with the specific cells on the tissue surface in deep.In addition, the excitation and emission spectra of these fluorescent dyes formerly verified makes it produce significant background noise, makes target tissue be not easy to be detected.In addition, as what discuss in above-mentioned background part, there is low shelf life stability shortcoming in the dyestuff based on fluorescence.EC17 as the thiourea bridge in this compound instable result and be easy to decompose.In addition, owing to EC17, the defect fluorescein of the relative high levels non-specific background noise had from the collagen protein in the surrounding tissue of imaging position is used.In addition, biological chromophore, particularly hemoglobin absorption visible ray further limit the serviceability of the dyestuff mixing fluorescein.This means that conventional dyestuff is not easy to detect and bury the tumor that the degree of depth in the tissue exceedes several millimeters.In addition, from the fluorescence of fluorescein under low pH (lower than pH5) by quencher.

In order to make dye substance for detection and guided operation or provide other imaging of tissue, importantly overcome these defects.

Comprise in the conjugate of nir dye in preparation and consider several standard.Be easy to synthesis and chemical stability be main chemical attribute.Consider spectral characteristic, such as Absorption and emission spectra and quantum yield.Have rated several biological nature, such as, binding affinity in cell research, use have the imaging of whole body animal and the bio distribution of the mice of tumor.Consider chorologic several aspect especially, comprise each oral distribution dead mouse, survival mice imaging and dosage escalation after 2 hours.Finally, take safety and consider, comprise maximum tolerated dose (MTD), immunohistochemistry (IHC) is analyzed and general Clinicopathological analysis.

The application provides the pteroyl-conjugate of nir dye, its be stable, infra-red range send fluorescence and the deep penetrated in target tissue to carry out specificity and brightness qualification to expressing the tissue regions of folacin receptor.More specifically, described pteroyl-conjugate is connected to nir dye by aminoacid connection base.Even more specifically, have been found that then the fluorescence intensity of dyestuff is maintained and even is enhanced if it is tyrosine or tyrosine derivative that aminoacid connects base.

Aminoacid is defined as to comprise and is connected to carboxylic acidfunctional group aminefunctional group and have specific for every seed amino acid side chain.Alpha amino acid is general formula R ⁵cH (NH ₂) any compound of COOH (a-amino acid), wherein R ⁵be selected from H or amino acid side chain known arbitrarily.

Beta amino acids is defined as comprise and is connected on β carbon aminefunctional group and being connected on α carbon carboxylic acidfunctional group.β homoamino acid is defined as to comprise and is connected on β carbon aminefunctional group, to be connected on α carbon carboxylic acidfunctional group and initial from α or β carbon side chain, wherein said side chain is combined with another kind of aminoacid.

Naturally occurring aminoacid can be divided into following 4 classes: (1) acidic amino acid, (2) basic amino acid, (3) center pole acidic amino acid and (4) neutral non-polar amino acids.Representational aminoacid in these are different classes of such as, including, but not limited to (1) acid (electronegative) aminoacid, aspartic acid and glutamic acid; (2) alkalescence (for positive charge) aminoacid, such as arginine, histidine and lysine; (3) neutral polar amino acid, such as glycine, serine, threonine, cysteine, tyrosine, agedoite and glutamine; (4) neutral non-polar (hydrophobicity) aminoacid, such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine.

Amino acid whose conservative replacement in naturally occurring aminoacid sequence can be selected from other member of naturally occurring aminoacid generic.Such as, amino acid whose aliphatic lateral chain is glycine, alanine, valine, leucine and isoleucine.The conservative replacement of naturally occurring amino acid valine comprises use glycine, alanine, leucine or isoleucine.

Amino acid whose aliphatic-hydroxyl side chains base is serine and threonine.The amino acid whose side chain radical comprising amide is agedoite and glutamine.Amino acid whose aromatic side chains base is phenylalanine, tyrosine and tryptophan.Amino acid whose basic side chain base is lysine, arginine and histidine.The amino acid whose sulfur side chain radical that comprises is cysteine and methionine.The example that natural conserved amino acid replaces is: valine for leucine, serine for threonin, phenylalanine for tyrosine, lysine for arginine, cysteine replace methionine and asparagine for glutamine,

In preferred embodiments, the tumor cell of this pteroyl-conjugate specifically in target tissue is confirmed herein.In addition, fluorescence intensity is greater than other intensity observed in advance for the polymeric dye of the folate-targeted of folate receptor-positive tumor of use.The intensity of this increase allow targeting and clearly identify from monitor the zonule (such as less tumor) of the biological sample of tissue.In addition, the intensity of the increase of the compound of the application provides additional advantage, namely can use the fuel compared with low dosage/consumption and still produce significant result.Therefore, the compound of the application produces more economical imaging technique.In addition, there is additional advantage, namely the compound of the application is lower than dosage with conventional imaging Compound Phase, still can will use the subsidiary toxicity of exterior materials to health and other side effect is minimized.

In addition, identify that little tumor causes more accurate and more effective to the excision of the primary tumor not producing edge and can identify and remove the lymph node of hiding metastatic carcinoma cell exactly and identify satellite disease.These advantages are actively correlated with to the better clinical effectiveness of treated patient separately.

In particular embodiments, find that the aminoacid of non-tyrosine causes polymer fluorescent to lack as the application connecting base.Such as, see the discussion of reaction scheme I.Pay particular attention to, route of synthesis produces less desirable by-product 4 as primary product, and it does not have NIR characteristic.

But pay close attention to except tyrosine and tyrosine derivative, the pteroyl-conjugate with the nir dye of cysteine or cysteine derivative may be useful.In addition; pay close attention to pteroyl-or folic acid moieties is directly connected with dyestuff or dyestuff and pteroic acid or folic acid are connected base by amine and connect the fluorescence intensity also created from compound and lack, and between pteroyl-(targeting moiety) and nir dye (fluorescing fractions), to there is tyrosine or tyrosine derivative as coupling part be useful for the fluorescence maintained or strengthen conjugate compound.The compound based on tyrosine of the application is connected base without the need to extra amine and is connected to compound S 0456 and also causes Fluorescence Increasing because put together to be puted together by the phenol moieties of tyrosine.

Use together with point daughter layer angiographic imaging system that described compound can mediate with fluorescence, such as, for detecting those of near-infrared fluorescent activation design in deep tissue.Described compound provides molecule and tissue specificity, the high fluorescence contrast of generation, brighter fluorescence signal and reduces background autofluorescence, thus the earlier detection of proper troubles diseased tissues (such as cancer) and molecular target evaluation are improved.Described compound may be used for deep tissue three-dimensional imaging, targeting operation and for quantitative to the amount of the target cell type in biological sample.

Compound

In one aspect, the application relates to the compound of contained (I):

Wherein:

X is aminoacid or derivatives thereof, and

Y is the dyestuff near infrared range with fluorescence excitation and emission spectra, and described compound maintains or strengthens the fluorescence of Y.

In some embodiments, in aminoacid or amino acid derivativges photoinduced electron emission spectra, electronic or electronic emission spectrum and electronic relative to the displacement of the electronic spectrum of unmodified dye molecule.Compatibly, displacement in electronic spectrum is red shift (namely to longer wavelength/lower frequency displacement), and this contributes to improving the detection of compound in near-infrared (NIR) spectrum window width and/or reduces the amount of background signal, autofluorescence, interference from developed region surrounding tissue.More specifically, the NIR dyestuff comprising the electronegative atom being incorporated to 6-ring is used to observe displacement in electronic spectrum especially.Therefore, in some embodiments, aminoacid or aminoacid (X) derivant comprise the part being rich in electronics, such as oxygen, sulfur or nitrogen.This amino acid whose example can comprise cysteine, methionine, threonine, serine, tyrosine, phenylalanine, tryptophan, histidine, lysine, arginine, aspartic acid, glutamic acid, agedoite and glutamine or derivatives thereof.

In embodiment in this, the application provides the compound of formula (I) a, (I) b, (I) c and (I) d:

Wherein Tyr, Cys, Ser and Lys group represent respectively tyrosine, cysteine, serine and lysine amino acid residue or and derivant, and L is preferably pteroyl-or folic acid, and each self-contained independent optionally non-existent solubilizing group selected of Rx

Wherein Tyr, Cys, Ser and Lys group represent respectively tyrosine, cysteine, serine and lysine amino acid residue or and derivant, and L is preferably pteroyl-or folic acid.Preferred L is pteroyl-.

In particularly preferred embodiments, the application provides the compound of formula I (a), and wherein Tyr is selected from:

Compatibly, compound disclosed herein at about 650nm-1000nm, such as and preferably in the near infrared region of about 800nm, there is maximum light absorption wavelength.

In particularly preferred embodiments, compound disclosed herein comprises part (L), and it effectively makes described targeting compounds to specific cells or organization type and allows the cell or tissue imaging to this targeting.Preferred L is pteroyl-part or folic acid moieties and more preferably L is pteroyl-part.But, pay close attention to those skilled in the art and some other ligand L can be used to make described targeting compounds to the specific cells surface protein paid close attention to or receptor protein.In concrete and preferred embodiment, described part comprises pteroyl-:

The synthesis of compound

Compound disclosed herein can use known in the literature conventional method to prepare, see, such as, dye composition as described in the synthesis of above-mentioned report.

But in particularly preferred embodiments, the application is provided for the more effective synthetic method generating compound described herein (i.e. the compound of formula I).Such as, can according to the general reaction scheme of summarizing in following reaction scheme I, II and III, preparation has the compound of formula I (a)-I (d).

Reaction scheme I example is in advance for the synthetic reaction route of the compound of production I, and wherein target ligand comprises the folic acid being connected to dye molecule by aminoacid (lysine).In brief, the ether derivant by being connected to the bridging of amino acid whose amido modified folate ligand and dyestuff reacts under the condition producing product (3) and (4).But it should be noted that compound 3 is the compounds preferably expected, and synthesis path causes there is less desirable by-product 4 as primary product, it does not have NIR characteristic.In addition, its spectral characteristic is that pH is dependent.Therefore, this reaction scheme shows the major defect of ether bridged dyestuff.In the conventional production practices of these dyestuffs, the yield of 30-60% is the yield of the product expected, and the yield of 40-70% is the yield of less desirable by-product.

Reaction scheme II provide synthesis by way of, it comprises only 3 reactions steps and provides product Compound (5) (higher than 98%) of high yield.In brief, by targeting part (1), (example is in reaction scheme II, with pteroyl-) and optionally comprise protecting group to avoid less desirable aminoacid with the reaction of the group of the amino of non-amino acid or amino acid derivativges (2) at HATU [(O-(7-azepine benzo triazol-1-yl)-N, N, N ', N '-tetramethylurea hexafluorophosphate)] mix in/DIPEA (diisopropylethylamine)/DMF (dimethyl formamide) solvent system, in room temperature reaction enough time (5 minutes), to make (2) by amido functional group and part (1) coupling, obtain (3).Can by diluted acid being added in this reactant mixture to advantageously compound precipitates (3).More specifically, with 1NHCl (hydrochloric acid) compound precipitates 3, obtain finalization compound, purity, more than 98%, in these embodiments, avoids and uses expensive HPLC or column chromatography steps.By making compound at room temperature at TFA (trifluoroacetic acid): in water: TIPS (tri isopropyl silane) solvent system, reaction makes compound (3) react to remove the protecting group on the amino acid moiety of compound, obtains compound (4).By with ether or methyl tertiary butyl ether(MTBE) deposition and purification compound 4, obtain the purity more than 98% when not carrying out HPLC (high performance liquid chromatography) or column chromatography.Make compound (4) at aqueous alkali system (such as NaOH; sodium hydroxide) middle reaction; to remove protecting group functional group; in water, at 80-100 DEG C of temperature, 15 minutes are reacted with dyestuff (S0456) subsequently when slightly molar excess; to allow dyestuff and (4) coupling, obtain finalization compound (5).With acetone precipitation compound 5, obtain the Pte-Tyr-S0456 pure more than 98%.When naoh is used, the sodium salt of Pte-Tyr-S0456 is produced.

Reaction scheme II:

Reaction scheme III provide produce compound disclosed herein optional solid phase synthesis by way of and provide and such as similar described in reaction scheme II yield.In brief; the aminoacid (in following reaction scheme III, example is the protected tyrosine being connected to resin bead) combined with substrate (1) is made to react to remove Fmoc (fluorenylmethoxycarbonyl groups) protecting group in the DMF solution of 20% piperidines; react in HATU/DIPEA/DMF with targeting part (other example is pteroyl-hereafter) subsequently; its response time and temperature are enough to make this part and amino acid whose amine functional group coupling, obtain (2).Compound (2) is reacted so that at TFA in series reaction: water: remove any protecting group on substrate and aminoacid in TIPS solvent system, obtain (3).After the similar final step such as described in reaction scheme II; compound (3) is reacted in aqueous alkali system; to remove protecting group functional group; react in water with dyestuff (S0456) when slightly molar excess subsequently; its response time and temperature allow dyestuff and (3) coupling, obtain finalization compound (4).

Reaction scheme III:

Above-mentioned reaction scheme is several non-limiting synthetic method of example only, can prepare compound disclosed herein by these methods.It will be understood by those skilled in the art that and to above-mentioned reaction scheme qualification and can be incorporated to modification, these modification can provide the other compound of the physical characteristic with the application's scope.Such as, although above-mentioned reaction scheme example folic acid and pteroyl-are as the targeting part of compound disclosed herein, it will be understood by those skilled in the art that and be easy to other targeting part is incorporated to synthetic reaction route and the optional compound of production I.As another example, it will be understood by those skilled in the art that can by adjusting the length of polymethine chain and selecting applicable aryl or heteroaryl (such as indole and benzindole) and be connected absorption/emission wavelength that amino acid group regulates the dye moiety of compound.In another example, those skilled in the art approve, extinction coefficient and the fluorescence intensity of dyestuff can be changed by adjustment polymethine chain rigidity known as this area (such as, such as by ring system is introduced polymethine chain, cyclohexene, cyclobutane ketone etc.).Therefore, those skilled in the art can synthesize to prepare any compound disclosed herein and the particular physical characteristics that can change this compound by selecting the reagent be applicable to change.

Using method

Just as noted, demand is existed for targeting specifically to the nir dye compound in in-house region.Namely described compound may be used for imaging technique and the Diagnosis and Treat intervention of aided disease.As what discuss in detail above, compound provided herein is used as the preparation in dyestuff and NIR light spectrum district.Like this, described compound is widely used in numerous imaging, diagnosis and targeted therapies.

In particular embodiments, the application relates to the method for at least one (such as the compound of formula I, I (a), I (b), I (c) and/or I (d)) being incorporated to compound disclosed herein, and these compounds may be used for specifically and tumor delicately in appraisement organization.More specifically, identified tumor can be excised by operation method therapeutic immediately.In this manner, the compound of the application can use the excision guided in fluorescence of tumor, lymph node etc.Or the compound of the application is easy to for whole body imaging, wherein by described compound administration in experimenter and fluorescence localization be conducive to identify tumor locus.

In this manner, the compound of the application may be used for the interior qualification of body of the illing tissue of the experimenter having this to need.The method of the application comprises and is used in about 600nm-and is about body part in body that illumination that 1000nm near infrared range has an at least one excitation wavelength penetrates the experimenter comprising illing tissue.Make to derive from and be applied to experimenter and the position and/or the surface area that directly manifest the illing tissue determining experimenter specifically in conjunction with the illing tissue in body part and/or the Fluorescence Fluorescence of compound that is absorbed the application as the response at least one excitation wavelength.

The light with 600nm-850nm wave-length coverage belongs to the near infrared range of spectrum, compares with visible ray, and visible ray belongs to the scope that about 401nm-is about 500nm.Therefore, exciting light for the diagnostic method implementing the application comprises the light of at least one wavelength to irradiate tissue in infrared waves strong point, thus excites compound so that the fluorescence obtaining the region of the compound of self-absorption the application clearly manifests and is different from the autofluorescence of surrounding tissue.Exciting light can be monochromatic or polychrome.In this manner, the compound of the application is favourable, because they eliminate the demand for using strobe utility, described strobe utility may be used for the diagnostic image obtaining expecting, as long as fluorescent probe sends fluorescence at the wavelength place lower than about 600nm.In this manner, the compound of the application avoids fuzzy diagnostic image, these fuzzy diagnostic images be as can from health tissues reflect and the exciting light of the wavelength causing fluoroscopic image resolution to lack resultant.

Can be equipped with overhead light to surgical operation operating room, these overhead light produce the optical wavelength in the optical emission spectra of the diagnostic method for implementing the application, such as, produce the lamp of the light of applicable wavelength.This light only may be used for by switching to other light (to eliminate the visible exterior light that may reflect from the tissue of body part under study for action) in situations in the surgery room and the exciting light of near-infrared wavelength being shining into body cavity or implementing the diagnostic method that open surgery implements the application, and the fluoroscopic image that the eye of observer's (such as surgeon) is directly received mainly derives from the fluoroscopic image of the fluorogen in the visual field.The light deriving from the source of 600nm-850nm scope, preferably 750nm-850nm scope may be used for directly being reached by observer the target of development, make to reflect from body part but not light from the part of emitting fluorescence be reduced to minimum or be eliminated.

Therefore, in the diagnostic method of this instrument text, make illing tissue (with combine or the targeting construct that absorbs) " exposures " in exciting light (such as by the open zone of operation generation or the endoscopic delivery of light to interior location.The disclosed method of the application is openly particularly suitable for the body detection of the illing tissue be positioned on the body position of experimenter, such as in natural body cavity or in the open area that generates of operation, wherein illing tissue be " smooth view " form (being namely exposed to human eye) be conducive to biopsy operation or excision because absorbing the compound of the application the region that highlights.Because the exact position of tumor tissues and/or surface area are determined easily through the absorption of the compound of the application, so use method valuable guide for surgeon provides of the compound of the application, this surgeon needs " observation " in real time to the definite profile, size etc. of lump, to be excised in operation is carried out.

Therefore, in particular embodiments, this application claims by making described contact tissue comprise the compositions of the compound (compound of such as formula I) of the application and allow the compound in compositions to distribute in tissue and with folacin receptor position occur the interactional time to carry out optical imagery to the biological tissue of expressing folacin receptor.In this interaction after time enough, send fluorescence with excitation light irradiation tissue to cause the compound in compositions.Then and wherein observe this fluorescence if, be then the region comprising folacin receptor by this fluoroscopic examination.

In a similar way, the compound of the application is for the identification of the target cell type in biological sample, and by making biological sample contact this compound to carry out, its time of contact and this compound of conditions permit are in conjunction with at least one cell of described target cell type.Then optical detection is carried out for the compound combined, make the fluorescence deriving from the near-infrared wavelength of the target compound of the combination of the application existed represent that target cell type is present in biological sample.The method provides the image of the acell type of targeting in evaluated tissue thus.Most preferably, described acell type is the lymph node that tumor cell or tumor cell have diffused to.

These methods advantageously provide carries out improving one's methods of the operation of image guidance to experimenter, because compositions that the condition at given surgical position and time uses the compound comprising the application is developed contributing to surgeon to tissue to be removed to be enough to make compound to be accumulated in.Preferably, described tissue is tumor tissues, and makes the compound of described tissue resorption send light to be conducive to by the near-infrared fluorescent of compound, to use infrared light to make tumor imaging.By means of the development that the targeting compounds of the application promotes to tumor locus, excision by infrared ray excited time send fluorescence region can improve and accurately go out even little tumor.

Should be appreciated that the application operation method any number of in, can before carrying out excision so that above-mentioned expose operation chamber and tumor locus after use them.

Position in the body that if the disease sites of presumption is natural body cavity or operation to be produced, then can optionally endoscope apparatus be used for exciting light to be delivered to this position, the fluorescence at receipt source this position in body cavity and the auxiliary through image from the fluorescence of illing tissue be formed.Such as, the camera lens in endoscope apparatus can focus on the fluorescence of detection, is formed because contribute to image.Think that the fluorescence of this endoscopic delivery used herein " is directly observed " by doctor, and targeting oligomer combine or wherein absorb it tissue for endoscope's necessarily " the smooth visual field " form, because for the light of the diagnostic method of the application not containing the optical wavelength penetrating tissue, the wavelength of such as near infrared range.Or, can by arbitrarily easily mode exciting light imported body cavity or comprises the operation open area of the targeting construct used as described herein, and consequent fluoroscopic image can without from endoscope auxiliary under observed directly by the eye of observer.Use or do not use endoscope apparatus auxiliary from any type, the fluoroscopic image produced by the application's method make it possible to do not have image processing device auxiliary under observe this fluoroscopic image, described image processing device is CCD camera, TV detector, photon collection device etc. such as.

Pay close attention to the diagnosis of the application or formation method can make surgeon/doctor by open surgery observe simultaneously/observe/show ill or abnormal structure, to be conducive to biopsy operation or excision.Because the position of illing tissue and/surf zone measure easily through the diagnostic method of the use compound as herein described of the application, so the method for the application is valuable guiding for surgeon, described surgeon needs the definite profile, size etc. of knowing lump, so that the excision when operation is carried out.Especially, notice that the compound of the application sends intensity near infrared range and is greater than fluorescence described in advance.Like this, advantageously, pay close attention to this less compound of needs and realize diagnosing image.In addition, the compound of the application can penetrate tumor and thus the application can advantageously provide removing tumor larger degree of accuracy.

The application is provided in the method in operation process, the experimenter needed being used to diagnostic operation, by using the compositions of the compound comprising the application to this experimenter and using up body in the body that irradiates and comprise the experimenter of illing tissue and assign to carry out, the light used has at least one wavelength of about 600nm to about 850nm scope, thus the illing tissue that can directly show in body part described in the specific binding that derives from and be applied to experimenter and/or the fluorescence of targeting construct be absorbed, wherein said targeting construct sends fluorescence as the response at least one excitation wavelength, thus position and the/surface area and remove at least part of tumor tissues of the illing tissue of experimenter can be measured.

In another embodiment, the application is provided for the method for the tumor tissues of the experimenter needed being carried out to in-vivo diagnostic.In this embodiment, the method for the application comprises the compound making to contact the multiple labelling detected in vitro from the tumor cells specimens of described experimenter, and they are separately in conjunction with different tumor type or be optionally absorbed; Deterministic compound combines with it or by sample tumor Cell uptake; Use at least one biocompatibility targeting construct of diagnosis effective dose, it comprises determines and sample tumor Cell binding and/or the compound of the application be absorbed and the fluorogen about 600nm being had to response at least one optical wavelength of about 850nm scope; Position and/or surface area with tumor tissues in body part in diagnosis body, directly shown by the fluorescence making to derive from tumor tissues the targeting construct combining or be absorbed when penetrating with the illumination providing fluorescent target at least one excitation wavelength of construct and carry out.

In some embodiments, the fluorescing fractions of single type depends on the fluorescence (namely from conjunction with illing tissue or the fluorescent target that is absorbed to construct) that generates from the body part irradiated and is the light source that described targeting construct contacts near infrared spectrum.

In other embodiments, pay close attention to multiple (namely two kinds, three kinds, four kinds or multiple) targeting construct to be used for obtaining diagnostic image.This other targeting construct can be the other compound being different from the first such the application.Or described other targeting construct can comprise dyestuff as herein described, but pteroyl-part is substituted by the part of the another kind of receptor of non-folate receptor.In other embodiments, described other targeting moiety can be other the targeting construct (the such as antibody or its bioactive fragment that send fluorescence, it connects fluorogen), they are in conjunction with the tumor of imaging or other receptor structural or antigen (such as the position of atherosclerosis, infection, cardiovascular disease, neurodegenerative disease, immune disease, autoimmune disease, respiratory system disease, metabolic disease, genetic diseases, infectious disease, osteopathia and environmental disease etc.).The targeting moiety other arbitrarily of selectively targeted tumor or structural specific part can be used, as long as it has specificity for monitored position.The object sending the targeting construct of fluorescence is in addition to increase the intensity of fluorescence on monitored position, contributes to thus detecting the ill or abnormal structure in body part.Such as, specify tumor can have a large amount of labelling, and except the compound of the application, the mixture of fluorescing fractions is also provided, it has specificity to appointment tumor, the tissue site that the signal deriving from this tumor is paid close attention to by more than one targeting and be positioned the compound at this position or fluorescing fractions generates.

In fact, those skilled in the art the mixture of detecting portion can use the compound of the application and any target tissue that these compounds and targeting moiety can be made to combine may reside on research position and/or be absorbed individually or as targeting, and then provide light source to supply.Typically, compound and the targeting moiety other arbitrarily of the application can use certain hour before surgery, and can be used certain hour at the fluorescent chemicals and fluorescence construct other arbitrarily allowing the application by the composition forms that target tissue absorbs.

Those skilled in the art can design the combination sending the targeting construct of fluorescence of using successively, and they separately can specifically in conjunction with target site.The all targeting constructs for the identification of target tissue sending fluorescence being preferred for this mixture comprise with the application's Compound Phase co-wavelength band in or the fluorogen (such as fluorescence being sent for the near-infrared wavelength sensitivity in the compound of the application) at phase co-wavelength place, needs are used for exciting the Different Light quantity of the fluorescence from all different targeting constructs being used for implementing the application's method to reduce to minimum simultaneously.But the other targeting moiety paying close attention to not this Applicant's Abstract graph compound can send the response of fluorescence as the irradiation light (namely having different wave length) to the color different from the fluorescent chemicals of the application.The color distortion deriving from the compound of the application and the fluorescence of other target compound can contribute to position and the size that observer determines illing tissue.In some instances, be desirably in the compound that targeting to the targeting construct of target normal structure comprises the application of fluorogen and targeting illing tissue, the contrast between illing tissue and normal structure be enhanced further to contribute to position and the size that observer determines target tissue.Except the compound that the disclosure is text, the application of this other fluorogen and targeting agent provides following advantage: any natural fluoresence deriving from normal structure is derived from the fluorescence of targeting to the fluorogen in the supplementary targeting construct of body part inner target tissue and shelters.The color distortion derived between normal and the fluorescence of target tissue is larger, then make the development Vietnamese side of the profile of target tissue and size for observer just.Such as, comprise to target tissue (i.e. abnormal structure) produce from the infrared light of the compound of the application fluorogen and observer contributed to the targeting construct sending fluorescence of fluorogen that health tissues produces green light distinguish target tissue and normal structure.Those skilled in the art are easy to the combination selecting the fluorogen providing different perceived color to contrast.

The spectrum of the light for implementing the application's method is selected to send the principal excitation wavelength of the part of fluorescence with the biocompatibility comprising at least one and be equivalent to comprise in targeting construct or targeting construct.Usually, the exciting light for implementing the application's method comprises at least one wavelength that about 600nm-is about the near-infrared wavelength light of 850nm scope.

But when the combination of targeting part sending fluorescence at different wave length place is for implementing in this application, the spectrum of exciting light must be wide to being enough to be provided for fluorogen used at least one excitation wavelength separately.Such as, when select the fluorogen of different colours with distinguish normal with illing tissue time, especially it is beneficial that light is that excitation spectrum comprises targeting to normally and the excitation wavelength of the fluorogen of target tissue.

As what notice herein, the compound of the application by as the pteroyl-of the part of the compound of the application or folate ligand specifically targeting to folacin receptor.Use in the embodiment of other targeting moiety wherein, select the targeting construct of this other targeting moiety to combine institute's concern target tissue and/or be absorbed specifically, the antigen such as, on the cell that combination is characterised in that disease in target tissue or abnormality or in it or other surface character.As in other diagnostic test, desired target to construct optionally in conjunction with target tissue or be absorbed or combine the antigen relevant to disease or abnormality; But, comprise the method that also may be used for implementing the application in conjunction with health tissues or cellularity or the targeting construct of ligand moiety that is absorbed, as long as the antigen concentration in the target tissue of the visual field or the affinity of targeting construct to target tissue are sufficiently more than health tissues, make the fluoroscopic image representing target tissue clearly can be shown as any fluorescence being different from and deriving from health tissues or structure in the visual field.

Such as, the feature of colon cancer is to there is carcinoembryonic antigen (CEA) usually, and this antigen is also in conjunction with some tissues in healthy individuals.But the concentration of CEA in carcinous colon is greater than the concentration found in health usually, therefore, anti-CEA antibody can be used as the ligand moiety in the application's enforcement.In another example, deoxyglucose is absorbed by health tissues and utilizes in various degree, and its metabolism in health tissues (except some known organs) such as heart is substantially lower than the metabolism in tumor.The known mode that deoxyglucose consumes in vivo may be used for assisting thus determines that those high-selenium corn that wherein deoxyglucose is unexpected send the region of the signal that there is tumor cell.

The disease that the application's method detects or abnormality can be any types, it is characterized in that there is known target tissue for known specific binding ligand.Such as, difference is cardiopathic is characterised in that for known specific binding ligand generation necrosis or ischemic tissue or produces atherosclerotic tissue.As another illustrative examples, the feature of breast carcinoma is to produce by for CA15-3, CA19-9, the cancerous tissue of the Identification of Monoclonal Antibodies of CEA or HER2/neu.Pay close attention to the feature of target tissue can be to produce known binding partner for surface antigen or the cell of born of the same parents' internal labeling (i.e. antigen) because many targeting constructs penetrate cell membrane.The representational morbid state that the application's method can be used to identify comprises such different syndromes such as such as dissimilar tumor, antibacterial, fungus and viral infection." exception " used herein tissue comprises precancerous condition, necrosis or ischemic tissue and the tissue etc. relevant to connective tissue disease and autoimmune disease.In addition, the diagnosis of the application method or the example of the type of target tissue that checks is suitable for using to comprise heart, mammary gland, ovary, uterus, lung, endothelium, blood vessel, gastrointestinal tract, colorectum, prostata tissue, endocrine tissue etc. and two or more combination arbitrarily thereof.

Merely as an example, the antigen of some common cancers and the body part usually finding them are well known by persons skilled in the art, and targeting part such as antibody or for these antigens antibody or in fact wherein antigen is the part of receptor is known in the art.Such as, CEA (cancer embryo can) finds usually in the tumor from colon, mammary gland and lung; PSA (prostate specific antigen or be sometimes called prostate specific membrane antigen (PSMA)) has specificity to carcinoma of prostate; CA-125 finds usually in the tumor in ovarian cancer source; CA15-3, CA19-9, MUC-1, estrogen receptor, progesterone receptor and HER2/neu find usually in breast carcinoma; Alpha-fetoprotein finds in testis and hepatic carcinoma; β-human chorionic gonadotropin finds in carcinoma of testis and choriocarcinoma; Estrogen receptor and progesterone receptor also find in uterus carcinoma tumor; And EGF-R ELISA finds usually in from the tumor of bladder cancer.Other tumor-targeting ligands and labelling are well known to the skilled person.In preferred embodiments, the application's use is used for folic acid or the pteroyl-part of targeting folacin receptor and makes dyestuff targeting to the PMSA targeting moiety of cancerous cell for targeting.

Concern can use dyestuff targeting as herein described these usually known tumor markers (by pteroyl-Partial Transformation being become the part of these labellings selectively targeted) arbitrarily; or, also and with the folacin receptor of the targeting compounds using the application combine these labellings of targeting.As discussed above, particularly advantageously specifying targeting moiety tumor had for several not isolabelings to be used as diagnosis mixture, wherein targeting is several is tagged to brighter and more clearly makes the degree of tumor imaging.

Except chemical combination beyond the region of objective existence, the targeting moiety in this mixture can also comprise protein or polypeptide, such as antibody or its bioactive fragment, preferred monoclonal antibody.Can also be for implementing the supplementary targeting construct of the application's method or comprise the polyclone or monoclonal antibody of using fluorogen labelling.Term " antibody " used in the application comprises complete molecule and functional fragment thereof, such as can in conjunction with the Fab of Epitopic determinants, F (ab') 2 and Fv.The method preparing these fragments be well known in the art (see, such as Harlow & Lane, Antibodies:ALaboratoryManual, ColdSpringHarborLaboratory, NewYork, 1988, be incorporated herein reference).As what use in this application, term " epi-position " refers to any antigenic determinant on the antigen that complementary antibody position combines.Epitopic determinants is usually made up of the chemically reactive surface grouping of molecule such as aminoacid or sugared side chain and usually has specific Three Dimensions Structure and specific charge feature.

Except antibody, described mixture can also comprise such compound, wherein be connected to fluorescent target and be selected from many biocompatible compounds to the ligand moiety of construct, it can bind receptor and/or preferentially absorbed by tumor cell and can be used as the ligand moiety in the targeting construct of the application specifically.Preferentially can be entered cell by the hydrophilic " window " etc. in surperficial or nuclear receptor (such as hormone receptor), hole, cell lipid bilayer by the compound of tumor cell " absorption ".

The example of such compound of target tumor is somatostatin, the somatostatin receptor-binding peptide class, deoxyglucose, methionine etc.Useful especially the somatostatin receptor-binding peptide class is the long-acting octapeptide analog of somatostatin, is called and presses down growth peptide (D-phenylalanyl-L-cysteinyl--L-phenylalanyl-D-tryptophanyl-L-lysyl--L-Threonyl-N-[2-hydroxyl-1-(methylol) propyl group]--L-half Guang amide ring (2 → 7)-disulphide); Lanreotide, namely presses down the oral formulations of growth peptide; P829; P587 etc.Somatostatin-binding peptide class is disclosed in US Patent No. 5,871, in 711, and make this peptide be covalently attached to radioisotopic method under the reducing conditions by its carboxyl-terminus amino acid to be disclosed in US Patent No. 5,843, in 401, these two sections of documents are intactly incorporated herein reference.Those skilled in the art prepare sensitive fluorescent the somatostatin receptor-binding peptide class for adopting this instruction by replacing in the part sending fluorescence of radiosiotope place the application.

When morbid state be neuroendocrine or endocrine tumors time, somatostatin and the somatostatin receptor-binding peptide class are used as the ligand moiety of the target tumor in targeting construct especially effectively.The example of the neuroendocrine tumor that the application's method can be used to diagnose comprises adenoma (produce GH and produce TSH), islet cell tumor, carcinoid, poorly differentiated type neuroendocrine carcinoma, minicell and nonsmall-cell lung cancer, neuroendocrine carcinoma and/or intermediate cell cancer, ovary, cervix uteri, endometrium, mammary gland, kidney, the neuroendocrine tumour of paranasal sinuses and salivary gland, meningioma, the tumor that well differentiated type colloid is derivative, pheochromocytoma, neurocytoma, ganglionic neuroblastoma, pheochromocytoma, papillary carcinoma in thyroid cell, papillary carcinoma and medullary carcinoma, Merkel cell carcinoma and melanoma and granuloma and lymphoma.These tumors known have the somatostatin receptor and somatostatin or somatostatin health binding peptide class can be used as the part targeting of the application's fluorescent target to the target tumor in construct.

For vasoactive intestinal peptide (Vasointestinalpeptide) (VIP) (I.Virgolini of VIP receptor scintigraphy art, EurJ.Clin.Invest.27 (10): 793-800,1997 also for the method for the application, to diagnose some endocrine tumorses of small-sized Primary Adenocarcinoma, hepatic metastases and gastrointestinal.

Can be deoxyglucose by another molecule example of the part of the target tumor of tumor preferential absorption, known its preferentially various dissimilar in absorbed.Deoxyglucose can be used to comprise melanoma, colorectum and pancreas tumor, lymphoma (HD and NHL), H/N tumors, ovarian cancer, breast carcinoma and the brain cancer (senior and pituitary adenoma), sarcoma (grade dependency), hepatoma, carcinoma of testis, thyroid (grade dependency) small cell lung cancer, bladder cancer and uterus carcinoma etc. as the tumor type that the part of target tumor detects.

The compound that may be used for other target tumor in the mixture of the application comprises 1-amino-Tetramethylene .-1-carboxylic acid and METHIONINE.METHIONINE is the necessary essential amino acids of protein synthesis.Known malignant cell changes methionine metabolism and needs the methionine of external source.

Specific binding tumor receptor and/or the other example of the compound of biocompatibility target tumor preferentially absorbed by tumor cell comprise mammalian hormones, particularly gonadal hormone, neurotransmitter and by tumor cells expression to link up by the compound of tumor cell preferential absorption each other, such as derive from chromosomal change such as clone in transfer or the new secretory protein construct of upset.

Hormone comprises gonadal hormone.Growth of Cells hormone, cytokine, endocrine hormone, erythropoietin etc. are also used as cancer target part fully.As is well known the art, many tumor types express the receptor of hormone such as estrogen, progesterone, androgen such as testosterone etc.This hormone is preferentially absorbed by tumor cell, such as, pass through specific receptor.

Can by well known to a person skilled in the art arbitrarily by way of using for implementing the targeting construct of the application's method and supplementary targeting construct, such as by local, intraarticular, pond, ophthalmic, ventricle be interior, in sheath, intravenous, intramuscular, intraperitoneal, Intradermal, tracheal strips, intracavity etc. and pass through their two or more combination in any any.

Optimally use the difference by way of the position according to treated morbid state or the doubtful disease diagnosed or tumor and change.Such as, in order to treat inflammatory disease and different tumor, the local application body part comprised by directly injecting excitation light irradiation is used, and (such as passing through intracavity) provides following advantage: can use targeting construct (such as fluorescently-labeled antibody) with high concentration, does not have possibility with the subsidiary complication risk of its systemic administration.

The targeting construct other arbitrarily that the compound and comprising can using the application with " effective dose " uses in the diagnosis mixture of the compound of the application is for diagnosis.Effective dose is any target tissue chemical development requisite targeting construct consumption of the body part contributing to the research making to be arranged in experimenter.Pay close attention to and comprise arbitrary mammal as term used herein " experimenter ", such as domestic pets, farm-animals or zoo animal, but be preferably people.Effectively for diagnostic application amount certainly according to the size of studied body part and position, targeting construct for the affinity of target tissue, the type of target tissue and use by way of difference and different.The local application of targeting construct typically needs the dosage being less than any systemic administration pattern, but, in some cases, the local concentration of targeting construct after local application higher than the concentration reached safely during systemic administration.

Because individual subjects may present the extensive change of symptom severity and often kind of targeting construct has the diagnosis ketone feature of its uniqueness, comprise targeting construct to the affinity of target, targeting construct by the clearance rate of body processes, the characteristic etc. of fluorogen that wherein comprises, so factor described in weighting is changed dosage by those skilled in the art thus.

Can according to known method, use the dispersant that is applicable to or wetting agent and suspending agent that the compound of the application and the mixture that comprises these compounds are mixed with sterile injectable suspension.Sterile injectable preparation can also be sterile injectable solution in the acceptable diluent of avirulent parenteral or solvent or suspension, such as, be at 1-4, the solution in butanediol.Aseptic fixing oil is commonly used for solvent or suspending medium.For this object, arbitrarily gentle fixing oil can be used, comprise synthesis monoglyceride class or diacylglycerol esters, fatty acid (comprising oleic acid), naturally occurring vegetable oil if Oleum sesami, Oleum Cocois, Oleum Arachidis hypogaeae semen, Semen Gossypii wet goods or synthctic fat vehicle are as ethyl oleate etc.Can mix buffer agent, antiseptic, antioxidant etc. as required, or they can comprise in the formulation.

Those skilled in the art are apparent, can carry out various change in this application when not departing from its scope, and thus the application except in this description particularly those disclosed off-lying sea comprise those as shown in accompanying claims.

There is provided embodiment hereafter only for the object of the specific embodiments of example the application, and not in advance to limit the scope of accompanying claims.As discussed in this article, differently can change the specific features of disclosed Compounds and methods for, what these different modes provided not necessarily has operability or advantage.Such as, compound can be incorporated to each seed amino acid and amino acid derivativges and targeting part, and this depends on the concrete purposes of compound used therefor.It will be understood by those skilled in the art that this modification is included in accompanying claims scope.

Embodiment

Embodiment 1: take tumor as the exploitation in the operation instructed at fluorescence of the nir dye of target

Complete excision malignancy disease is unique interference method reliably in cancer.Lamentedly, quantitative tumor resection is limited to the ability that surgeon is located all malignancy diseases and itself and health tissues distinguished usually.The operation that fluorescence instructs has been shown as the instrument of assisted surgery doctor qualification and removing malignant tumor lesions.Although verified non-targeted fluorescent dye is accumulated in some tumors passively, the ratio of the tumor obtained and background is usually poor and be difficult to the border determined between malignant tumor and health tissues.In order to prevent these problems, the application shows this affinity targeting part of exploitation, and it is combined in the receptor of overexpression on cancerous cell and sends the molecular selectivity of connection into these cells.

In the present embodiment, apply two kinds of tumour-specific targeting part (i.e. folic acid, its targeting folacin receptor (FR), near-infrared (NIR) fluorescent dye to be delivered to specifically the cancer expressing FR, only gives malignant cell obvious fluorescence thus.The NIR dyestuff of all FR-targeting that the present embodiment display checks combines the cancerous cell cultivated with low nanomolar range.In addition, when intravenous injection has the mice with metastatic disease of tumor, these identical part-NIR dye conjugates transmit fluorescence to the tumor tissues of expressed receptor, thus can excise expediently, and minimize the pollution of health tissues.

1A: materials and methods

The result shown in the present embodiment uses hereinbefore certain material and method to obtain.Pay close attention to those skilled in the art and can change these methods, reaction condition and test condition, and still produce the result of the usefulness of the NIR dyestuff of display the application FR-targeting.

A. the Synthesis and characterization of folic acid-NIR conjugate. all parts of the synthesis as reported in above-mentioned document be connected base.After purification, the NIR dyestuff making the part of targeting folic acid educate selection as shown in Fig. 1,5 and 8 is puted together.Use Reverse phase preparative HPLC [Waters, xTerraC1810 μm; 19x250mm; λ=280nm; Solvent Gradient: 0-30% or 80%B, 30min run, A=10mMNH ₄the water buffer (pH=7.0) of OAc, B=acetonitrile (ACN)] purifying dye conjugate.Use LC-MS (ESI) mass spectrography (Waters, X-BridgeC185 μm; 3.0x15mm) analyze the compound of purification.

The cultivation .L1210A cell of b. expressing the cell line of folacin receptor derives from doctor ManoharRatnam and KB cell derives from American Type Culture preservation center (AmericanTypeCultureCollection) (ATCC; Rockville, MD).Cultured cell in the folic acid deficiency 1640RPMI culture medium supplementing 10% heat-inactivated fetal bovine serum (HIFBS), 1%L-glutamine and 1% penicillin streptomycin (Invitrogen, Carlsbad, CA).In 5% carbon dioxide, 95% air wetting atmosphere, whole cell line is cultivated at 37 DEG C.

C. geometry affinity and the specificity of folic acid-NIR dye conjugate is analyzed by fluorimetry. KB (200,000 cells/well, 500 μ L) is inoculated into 24-well culture plate, makes it in 48h, form monolayer.With the fresh culture (0.5mL) of folic acid-NIR dye conjugate comprising progressive concentration under the existence of the doubly excessive competitive part of 100-and folic acid or its not in the presence of substitute the culture medium of the consumption in each hole.At 37 DEG C, incubation is after 1 hour, cell is rinsed with the fresh culture (3 × 0.5mL) being dissolved in 1%SDS aqueous solution (0.600mL), measuring fluorescence by being transferred to quartz cuvette pond, using AgilentTechnologiesCaryEclipse fluorescence radiation luminometric analysis at often kind of dyestuff maximum excitation and launching the fluorescent emission intensity removed.Dissociation constant (the K of conjugate is calculated by the schematic diagram using GraphPadPrism4.03 to draw the targeting nir dye concentration relationship of fluorescent emission unit and interpolation _d).

D. mice metastasis model in body. carry out all animals operation through releasing animal care from sufferings and applying committee (PurdueAnimalCareandUseCommittee) approval.In order to carry out involving the research of the tumor expressing FR, 5-6 age in week, female DBA/2 mice was purchased from HarlanLaboratories (Indianapolis, IN), will with in process, it to be placed on folic acid deficiency meals 2 weeks before carrying out each research.By using No. 30 syringe needles by 1x10 ⁶the left ventricle that L1210A (expressing FR) cell injects heart brings out tumor metastasis.Make tumor growth 4 weeks, after this, by intravenous to the NIR dyestuff being dissolved in the FR-targeting of the expectation of 100 μ l saline of animal injection 10nmols.After 4 hours, pass through CO ₂suffocate and put to death animal, imaging as described below.

E. there is the fluorescence imaging of the mice of metastatic disease. use the CaliperIvisLuminaIIImagingStation with LivingImage4.0 software to carry out animal imaging experiment.Setting for imaging AlexaFluor647 and DyLight680 conjugate: the grade of lamp: high; Excite: 605; Launch: Cy5.5; Epi throws light on; Frame method: (M) 4; FOV=7.5; F-stops=4; Acquisition time=1s.Setting for imaging DyLight750 and IR800CW conjugate: the grade of lamp: high; Excite: 745; Launch: ICG; Epi throws light on; Frame method: (M) 4, FOV=12.5; F-stops=4; Acquisition time=1s.

F. H & E that is normal and illing tissue dyes. and after imaging, anatomical organs, is stored in 5ml formalin, and feeding PurdueHistology & PhenotyingLaboratory carries out H & E and dyes.In brief, use SakuraTissue-TekVIP6 processed group tissue samples, use ThermoFinesseME microtome, with H & E reagent, use the dyeing of ShandonVari-Stain24-2 automatic staining device.Then the microscope slide video picture using the OlympusBH-2 research microscope with OlympusDP70 photographing unit to make H & E to dye.

B. result

A. be the synthesis of the NIR dyestuff of target with tumor. in order to selectivity cancer target, inventor makes the NIR that is purchased and folic acid put together.Synthesize most of folic acid-NIR dye conjugate of high yield, use HPLCs to be purified to evenly subsequently.

B. the binding affinity of targeting NIR dyestuff and specificity. because the load being connected to part can disturb ligand binding usually, so it is beneficial that test folic acid-NIR dye conjugate and the binding affinity of cancerous cell of expressing FR.Find that the binding affinity of all conjugates is at low nanomolar range (Fig. 2), wherein have some to change according to the difference of the dyestuff connected, the load that this enlightenment connects only moderately affects ligand binding.Also determine folic acid-NIR dye conjugate in vitro to the specificity of its receptor by adding excessive folic acid.As what observe in fig. 2, by incubation together with the folic acid of 100-times of molar excess close to inhibit combination quantitatively.

C. be the in-vivo imaging of the NIR dyestuff of target with tumor. before the tumour-specific evaluation of cancer target dyestuff in vivo, it is beneficial that compare the intensity of the dyestuff selected when being excited by tissue.In order to this object, the 1mL phosphate-buffered saline comprising 100nM and often plant dyestuff (AlexaFluor647, DyLight680, DyLight750, IR800CW) is put into Eppendorf tube, then under placing it in 1cm thickness fresh pig muscle section, what make under the same conditions to obtain in KodakImageStation and IVISLuminaImager organizes video picture, only selects the best to excite and emission wavelength all the time for often kind of dyestuff in often kind of instrument.IR800CW produces the brightest fluorescence signal, and DyLight750 produces the signal of intermediate intensity, AlexaFluor647 and DyLight680 shows the most weak fluorescence.

In order to the ability of metastatic tumor root nodule in more above-mentioned folic acid-NIR dye conjugate detection bodies, have developed neoplasm metastasis mouse model, it involves intracardiac injection 10 ⁶l1210A cell (expressing the cell of FR), then normally raises mice 4 weeks, grows to make primary tumor.Then treated the mice with tumor by tail vein injection with the folic acid-NIR dye conjugate that 10nmol selects, after 4 hours, euthanasia is implemented, for fluorescence imaging to mice.As what observe in Fig. 3, tumor locus is easy to distinguish, between fluorescence cancer root nodule and adjacent healthy tissues, produce sharp contrast.In some cases, even can observe fluorescent tumor (Fig. 3, upper group) in the image of intact mice, but, due to the difference of tumor size, position and the degree of depth, so NIR dyestuff can not be established clearly produce optimized image in intact animal.

Finally, in order to simulate great-hearted surgical environment, carrying out fluorescent tumor cutting tissue stage by stage, wherein first removing maximum lump, after the more obvious fluorescence lump of removing, excise less malignancy site (Fig. 4).Valuably, removing constitutional lump exposes Secondary cases metastasis usually, and it did not show before initial operation circulation, possibly cannot observe not having the auxiliary lower of tumour-specific fluorescence.After the excision of these multi cycle, when removing all visible fluorescences, carry out histologic analysis to the tissue of excision, these researchs disclose all fluorescence root nodules and are actually pernicious.Valuably, the stochastical sampling display non-fluorescence region of remaining tissue is nonmalignant (Fig. 4 d), and this enlightenment obviously eliminates cancer lesions quantitatively by means of the fluorescent dye taking tumor as target.

C. discuss

The operating second method instructed for fluorescence involves NIR dyestuff and tumour-specific targeting part puted together, and described part is removed from most of health tissues quantitatively in conjunction with cancerous cell energetically.The advantage of this method comprises: the i) fast velocity of tumor imaging, and this absorption owing to dyestuff and normal structure are removed and can launch this fact in several minutes of intravenous injection; Ii) stability of tumor imaging, this takes in this fact by cancerous cell by receptor-mediated endocytosis usually owing to part-dye conjugate; Iii) receptor targeted be do not exist, weak expression or cannot reach normal structure all has fluorescent specific; And iv) fluorescence " stream " do not existed from malignant tumor enters non-malignant tissue, this is owing to the high-affinity of part-dye conjugate to its receptor, produces the border that the height of clearly distinguishing cancer is determined.The shortcoming of this strategy derives from the following fact: the dyestuff of part-targeting has fluorescence all the time, even in discharge process, thus hinders the imaging of kidney and tumor of bladder, until the excretion of dyestuff is complete.

The result surprised from two people of these research generations is the malignant tumor lesions being easy to detect in vivo reduced size.Therefore, the more labor at several positions of the metastatic disease with puncture is disclosed, the optical instrument of high-resolution can be used to make the little cancerous cell bunch development to 50 μm.Because the cluster of even several cell finally can cause cancer return, so the ability detecting and remove even minimum metastatic lesion finally may cause the mortality rate of patient to reduce, thus deduction can design applicable photographing unit.

While finding that the operating major applications of fluorescence guidance is found sustainably, some application of this technology are concerned.First, can identify and excise more malignant tumor lesions because making tumor mass develop better.The second, protect normal structure to be (i.e. the brain cancer, breast carcinoma, cancer of pancreas, head and neck cancer etc.) in necessary information to greatest extent wherein, disappear modestly and scrape fluorescence infringement, until unstressed configuration retains, this tissue that can more effectively protect the health.3rd, the near-end lymph node being searched fluorescence infringement by laparoscopy finally can carry out by stages preoperative to cancer patient, thus gets rid of when clearly observing obvious metastasis for the demand of performing the operation and eliminate the demand for sentinel node operation sampling subsequently when single tumor mass only being detected.

In a word, the present embodiment display take tumor as the NIR dyestuff of target has transformation standard surgical procedures potential by the development improving malignant tumor tissue, thus causes removing illing tissue more completely and accurately and improving patient's prognosis.

Embodiment 2: there is height targeting affinity and the design of the near infrared fluorescent probe thing that the cancer operation best folic acid with sensitivity that fluorescence instructs is puted together and synthesis

Although use the same tumor qualification of complicated instrument, many malignant tumor root nodules still escape from detection, cause palindromia and usually cause death.According to the demand to the tumor qualification improved, introduce the new method for making malignancy disease develop in performing the operation.In first method, the fluorescent dye of quencher is injected with the animal of tumor through whole body, utilize the fluorescence optionally activated because of the quencher moieties that tumour-specific enzyme, pH change or oxidation-reduction potential changes release in malignant tumor block.In the second approach, fluorescent dye and tumour-specific targeting part are puted together, cause the dyestuff connected to be accumulated in the cancer of overexpression ligand receptor.Example for the cancer target part of the object after this comprises folic acid, the specificity of its display to folacin receptor (FR) positive cancer of ovary, kidney, lung, endometrium, mammary gland and colon.Valuably, in the operation of human ovarian cancer patient is carried out, test the fluorescent dye (folic acid-fluorescein or EC17) of folic acid-targeting in the recent period.In this study, by means of the fluorescent dye taking tumor as target eliminate higher than do not use it ~ malignant tumor lesions of more than 5X, confirm that the fluorescence infringement of all excisions is pernicious by pathology.

Lamentedly, use the major defect of above-mentioned clinical research to derive from the following fact: the dyestuff (fluorescein) of connection is transmitted in the fluorescence of visible range, namely wherein strong the and light of autofluorescence is difficult to penetrate tissue.Because the inducing self-body fluorescence and very more effectively penetrate tissue hardly of the light in near-infrared (NIR) district, so inventor's presumption, if NIR dyestuff is used to guide surgical operation, then tumor resection is possible more completely.But, existence be purchased NIR fluorogen limited amount and mainly based on the colored cyanines chemical constitution (Fig. 6) of being modified especially by each manufacturer.Fortunately, fluorogen series produces as reactive fluorogen separately, and it is easy to put together with paid close attention to protein or part, specifically for passing through targeting in chemical conjugate body.Major part is purchased experiment NIR fluorogen and obtains as N-hydroxy-succinamide (NHS) esters, and its fluorogen that may be used in N-terminal amine is puted together.In order to evaluate the NIR probe puted together with the best folic acid of the firm cancer surgeries for image guidance, inventor has made the functionalized folic acid of fast light stability NHS ester NIR dyestuff (following compound a-c) and N-terminal amine put together (Fol-EDA and Fol-Lys), obtains the NIR probe (1a-c and 2a-c) of expectation and main elimination by-product (1e-g and 2e-g).In order to the yield be improved, use more stable NIR dyestuff LS288NHS ester (d), NIR dyestuff 1d and 2d that the folic acid that separation has good yield is puted together.Characterize light performance and the light resistance of folic acid-NIR probe 1a-d and 2a-d of synthesis.

All folic acid-NIR probe 1a-d and 2a-d (500L of same concentrations; 5M, in PBS) fluorescence excitation and the almost similar intensity of emission spectra display.Cytotoxicity and the folacin receptor affinity of described probe is studied, at 5 groups with FR by In vitro cell experiment ⁺study in-vivo tumour targeting ability in the nude mice of KB tumor xenogeneic graft, also use LuminaII near-infrared fluorescence imaging systems inspection bio distribution.Lamentedly, the fluorescence intensity brightness of the bio distribution shown in the high yield mode of the probe folic acid-NIR synthesizing favourable Fig. 6 compound 1d and Fig. 7 compound 2d in tumor is lower than other folic acid-NIR probe 2 times of Fig. 6 compound 1a-c and Fig. 7 compound 2a-c.

According to described structure, useful change is at center vinylogy hexamethylene olefinic carbon (C (sp ²)) on by NIR dyestuff a-c phenol moieties replace (S _nR1), and the phenyl moiety on NIR dyestuff d is also with its corresponding final folic acid-NIR probe.These reasons above-mentioned have developed the folic acid-NIR probe of new modification owing to inventor, and it has light resistance, selectivity, susceptiveness and high fluorescent intensity at physiological ph.

In order to solve described problem, overall strategy is to select should comprise for deliquescent 4 SO as the S0456 being purchased precursor NIR fluorogen ₃h group is used for sunproof rigidity cyclohexene ring and the vinylogy cyclohexene chloride (C (sp being connected phenol oxygen with in the middle of fluorogen ²) Cl), this is of value to high sensitivity and the bright fluorescence of tumor imaging aspect in vivo.In addition, have studied topology requirement for the folacin receptor part of folic acid binding pocket to improve binding affinity and the selectivity of part and receptor.Not yet set up the crystal structure of folacin receptor, the folic acid fragment that namely pteroic acid lacks far-end glutamyl residue is disputed on in conjunction with this true existence enough good of high-affinity folacin receptor.In order to determine in glutamyl residue-carboxylic acid for whether useful in conjunction with folacin receptor, inventor has synthesized different aminoacids and non-amino acid pteroyl-conjugate and NIR probe thereof.Next, inventor compare the folic acid-NIR probe of all these new modifications with the external binding affinity of wild receptor positive carcinoma (cancel) cell and the in-vivo imaging (imaing) using folate receptor-positive KB tumor.Meaningfully, inventor does not observe the remarkable change of binding affinity, and in tumour-specific and fluorescence intensity, there is significantly change.This research is consistent with all folic acid-NIR conjugates and enlighten in glutamyl residue-and carboxylic acid is of value to tumour-specific targeting, absorption, and the center vinylogy hexamethylene olefinic carbon (C (sp in NIR dyestuff ²)) on phenol oxygen replace and be of value to the high fluorescent of tumor tissues.Therefore, need to research and develop simpler and more directly tactful in the folic acid-NIR fluorescent probe of the new modification of the best obtaining the cancer surgeries instructed for image urgently.Inventor's design and synthesize sensitive, fast light with folic acid-NIR fluorescent probe (pteroyl--Tyr-S0456 that the is new modification of tumor-selective; Fig. 6), it has the high fluorescent for clinical practice.

In the present embodiment, inventor's design have enhancing fluorescence intensity and sunproof take folacin receptor as the near infrared fluorescent probe (pteroyl-_ Tyr_S0456) of target.Display has the high targeting ability of the tumor of the folacin receptor-overexpression of bright fluorescence intensity.This new pteroyl-_ Tyr_S0456 conjugate improves the kinetics of described probe in mouse subject and the targeting ability enhanced the tumor of FR overexpression and susceptiveness.This new NIR probe of result display in the present embodiment has huge potential in diagnosis commitment tumor.

Embodiment 3

The relative analysis of OTL-0001 (FA-EDA-LS288), OTL-0002 (FA-EDA-IR800), OTL-0003 (FA-EDA-ZW800) and OTL-0004 (FA-EDA-Kodak2)

Materials and methods:

KB cell (people's nasopharyngeal cells system) derives from American Type Culture preservation center (Rockville, MD) and use containing folic acid comprise 10% heat-inactivated fetal bovine serum (AtlantaBiological, GA) and 1% penicillin streptomycin (Gibco, NY) 1640RPMI culture medium (Gibco, NY) at 37 DEG C at 5% carbon dioxide: make it as monolayer growth at least 6 generation in 95% air wetting atmosphere, then they are used for measure.

In athymic female nude mice (5 week age, 18-20g) at least 2 weeks purchased from Harlan (IN) and in the folic acid-shortage specific meal (Teklad, WI) being maintained gamma-irradiation, then start this research.Make animal reside in the bioclean of barrier with 5/cage to live away from home in framework.If need to give autoclaved tap water.Animal is made to reside in the time limit continuing this research in the gnotobasis of standard 12h fight-darkness cycle.Punched by ear and differentiate mice separately.Release animal care from sufferings and apply committee and have approved the operation of all animals.Animal care and research is carried out according to for the country of charitable animal process and international guidelines.

Whole body imaging:

To on 7 week age female nu/nu mice shoulder through subcutaneous vaccination KB cell (in without the RPMI1640 culture medium of folic acid 1.0 × 10 ⁶/ mice).Used caliper measurements tumor growth in vertical direction (using same scheme to monitor body weight) every 2 days, gross tumor volume is calculated as 0.5 × L × W ²(the most major axis of L=, and the axle that W=and L is vertical, in millimeter).Once tumor reaches 400 to 500mm ³volume, then inject the test article (FA-EDA-LS288, FA-EDA-IR800, FA-EDA-ZW800 and FA-EDA-Kodak2) in phosphate-buffered saline (100 μ L) of 10nmol to animal (2 mice/groups) through intravenous.After 2h, pass through CO ₂suffocate to euthanizing animals.Then the CaliperIVISLuminaIIImagingStation with LivingImage4.0 software (PerkinElmerInc, MA) is used to carry out whole body imaging (complete tumors) experiment.Imaging is arranged: the grade of-lamp: in; Excite: 745nm; Launch: 830nm; Epi throws light on; Frame method: 4 (M), FOV=12.5; F-stops=2; Acquisition time=1s.

Tissue biological distributes:

After whole body imaging, dissect animal, the fluorescence activity of the tissue (heart, lung, liver,spleen,kidney, stomach, small intestinal, large intestine, muscle, skin, tumor) using the analysis of IVIS imager to select as mentioned above.Imaging is arranged: the grade of-lamp: in; Excite: 745nm; Launch: 830nm; Epi throws light on; Frame method: 4 (M), FOV=12.5; F-stops=2; Acquisition time=1s.

Result:

Whole body imaging: as what observe in Fig. 7 a, FA-EDA-LS288, FA-EDA-IR800 and FA-EDA-ZW800 are mainly accumulated in folate receptor-positive tumor, essentially no fluorescence activity in other tissue.But FA-EDA-Kodak2 is not accumulated in tumor.In addition, the fluorescence intensity directly comparing the mice of the near-infrared IR dyestuff treatment of other folic acid of tumor fluorescence intensity ratio-put together of the mice of display FA-EDA-IR800 injection becomes clear (higher fluorescence intensity) (Fig. 7 b).

Conclusion:

From the best to the brightness of the most weak conjugate listed and specificity as follows: FA-EDA-IR800, FA-EDA-ZW800, FA-EDA-LS288, FA-EDA-Kodak2.Comprise the tumor accumulation fluorescence that the conjugate display of IR800 and ZW800 is the highest, and comprise the extremely low specificity of conjugate display to tumor of Kodak.The low Poison observed in Kodak conjugate may excite this true at 800nm owing to dyestuff.IVIS picture system does not have the filter excited at 800nm, and the low Poison thus in tumor can owing to using weak excitation wavelength.

Embodiment 4: the whole body imaging and the bio distribution that take folacin receptor as the nir dye of target

Materials and methods:

KB cell (people's nasopharyngeal cells system) derives from American Type Culture preservation center (Rockville, MD) and use containing folic acid comprise 10% heat-inactivated fetal bovine serum (AtlantaBiological, GA) and 1% penicillin streptomycin (Gibco, NY) 1640RPMI culture medium (Gibco, NY) at 37 DEG C at 5% carbon dioxide: make it as monolayer growth at least 6 generation in 95% air wetting atmosphere, then they are used for measure.

In athymic female nude mice (5 week age, 18-20g) at least 2 weeks purchased from Harlan (Indianapolis, IN) and in the folic acid-shortage specific meal (Teklad, WI) being maintained gamma-irradiation, then start this research.Make animal reside in the bioclean of barrier with 5/cage to live away from home in framework.If need to give autoclaved tap water and food.Animal is made to reside in the time limit continuing this research in the gnotobasis of standard 12h fight-darkness cycle.Punched by ear and differentiate mice separately.Release animal care from sufferings and apply committee and have approved the operation of all animals.Animal care and research is carried out according to for the country of charitable animal process and international guidelines.

Whole body imaging:

To on 7 week age female nu/nu mice shoulder through subcutaneous vaccination KB cell (in without the RPMI1640 culture medium of folic acid 1.0 × 10 ⁶/ mice).Used caliper measurements tumor growth in vertical direction (using same scheme to monitor body weight) every 2 days, gross tumor volume is calculated as 0.5 × L × W ²(the most major axis of L=, and the axle that W=and L is vertical, in millimeter).Once tumor reaches 400 to 500mm ³volume, then inject the test article in phosphate-buffered saline (100 μ L) of 10nmol to animal (2-3 mice/group) through intravenous.After 2h, pass through CO ₂suffocate to euthanizing animals.Then the CaliperIVISLuminaIIImagingStation with LivingImage4.0 software (PerkinElmerInc, MA) is used to carry out whole body imaging (complete tumors) experiment.Imaging is arranged: the grade of-lamp: in; Excite: 745nm; Launch: ICG830nm; Epi throws light on; Frame method: 4 (M), FOV=12.5; F-stops=2; Acquisition time=1s.

Tissue biological distributes:

After whole body imaging, dissect animal, the fluorescence activity of the tissue (heart, lung, liver,spleen,kidney, stomach, small intestinal, large intestine, muscle, skin and tumor) using the analysis of IVIS imager to select as mentioned above.Imaging is arranged: the grade of-lamp: in; Excite: 745nm; Launch: ICG (830nm); Epi throws light on; Frame method: 4 (M), FOV=12.5; F-stops=2; Acquisition time=1s.

Result:

Whole body imaging:

As what observe in Fig. 1, FA-EDA-LS288, FA-EDA-IR800 and FA-EDA-ZW800 are mainly accumulated in folate receptor-positive tumor, essentially no fluorescence activity in other tissue.In addition, bright (higher) (Fig. 2) of fluorescence intensity of the mice of the near-infrared IR dyestuff treatment of other folic acid of tumor fluorescence intensity ratio-put together of the mice of display FA-EDA-IR800 injection is directly compared.

Tissue biological distributes:

Under the same conditions by implementing euthanasia to every mice, take out its organ and use IVIS imager to carry out tissue biological's distributional analysis to its imaging to animal.As what observe in fig. 2, in FR-positive tumor and kidney, observe most high fluorescent.It is expection that kidney absorbs, since it is known the high-caliber folacin receptor of top film expression of the proximal tubule of kidney.In addition, possible situation is, probe is by renal excretion, and this is owing to its low-molecular-weight and half-life (most of folic acid conjugate has the half-life of <30min).

Conclusion:

From the best to the brightness of the most weak conjugate listed and specificity as follows: FA-EDA-IR800, FA-EDA-ZW800, FA-EDA-LS288, FA-EDA-Kodak2.Comprise the conjugate display the highest tumor accumulation fluorescence of IR800 and ZW800, and the conjugate display comprising Kodak is to its low Poison compared with other of the extremely low specificity of tumor.

A. materials and methods

Materials and methods as herein described is used to obtain the result shown in the present embodiment.Pay close attention to those skilled in the art and can change these methods, reaction condition and test condition, and still produce the result of the usefulness of the NIR dyestuff of the FR-targeting of display the application.

A. the Synthesis and characterization of folic acid-NIR conjugate. Synthesis and characterization folic acid NIR conjugate as described in example 1 above substantially.

B. folic acid-NIR conjugate (4) is to the RA of FR. and the KB cell of overexpression FR-α is seeded on the Falcon culture plate of 24-hole (100,000 cells/well), makes it within the 24h time limit, form monolayer.Under the test article of progressive concentration (0.1nM-1 μM) or the existence of folic acid fresh culture (0.5mL) middle 10nM [ ³h]-folic acid substitutes the culture medium consumed in each hole.At 37 DEG C of incubations after 1 hour, rinse cell to remove any unconjugated active material with PBS (2x0.5mL) and 1M trichloroacetic acid (1x0.5mL).After being added on the sodium lauryl sulphate (0.5mL) of in PBS 1%, cell is proceeded to the scintillation vial of each self-contained Ecolume scintillation cocktail (3.0mL), in, count with a. Liquid Scintillation Analyzer.Use the schematic diagram of the radioactivity of Cell binding and test article concentration relationship, apply GraphPadPrism4 and calculate RA.

C. the mouse model of subcutaneous tumor xenogeneic graft in body. on 5 week age female nu/nu mice shoulder through subcutaneous vaccination KB cell (in RPMI culture medium 1.0 × 10 ⁶/ mice).Used caliper measurements tumor growth in two perpendicular direction (using same scheme to monitor body weight) every 2 days, gross tumor volume is calculated as 0.5 × L × W2 (L is most major axis, and W axle is perpendicular to L, and unit is millimeter).Once tumor reaches 400 to 500mm ³volume, being then used in phosphate-buffered saline (100 μ L) is that the NIR dye conjugate (10nmol) of target treats animal with folacin receptor.After 2h, pass through CO ₂suffocate to sacrifice of animal animal, imaging as described below.

D. there is FR ⁺the fluorescence imaging of the mice of KB tumor. use the CaliperIVISLuminaIIImagingStation with LivingImage4.0 software to carry out animal imaging experiment.For making the setting of AlexaFluor647 and DyLight680 conjugate imaging: the grade of lamp: high; Excite: 605; Launch: Cy5.5; Epi throws light on; Frame method: (M) 4, FOV=7.5; F-stops=4; Acquisition time=1s.For making the setting of DyLight750 and IR800CW conjugate imaging: the grade of lamp: high; Excite: 745; Launch: ICG; Epi throws light on; Frame method: (M) 4, FOV=12.5; F-stops=4; Acquisition time=1s.

B. result

A. be the synthesis of the NIR dyestuff of target with tumor. in order to selectivity cancer target, the dyestuff that inventor uses NHS to activate or Williamson ether synthetic reaction, use the chloro-derivant of NIR dyestuff to make to be purchased NIR dyestuff to be puted together by amido link with folic acid or pteroic acid salt.Pte-aminoacid-NIR, especially tyrosine has been synthesized with high yield (>98%), do not use any HPLC or special purification technique (by precipitation), and very high purity (>98%).The aminoacid identified herein is tyrosine and analog, cystine, serine, lysine etc.NIR dyestuff used is S0456, Kodak, S0121 and S2076 (being not limited thereto).But, the synthesis of ether bridged NIR conjugate such as folic acid-IR800CW (3) and the folic acid-ZW800 (5) of folic acid causes producing a large amount of by-product (Figure 10 B), it needs special purification technique, such as HPLC, causes production cost higher thus and adds the time limit of preclinical to clinical conversion.This not only affects the progress of cancer surgery, and the patient of novel treatment is waited in impact.In addition, higher production cost may affect indirectly patient and Insurance providers thereof, and this cost owing to medicine increases.Importantly, Pte-Tyr-S0456 reaction does not produce any less desirable by-product, and back-pressure completes in 15min, and yield is high and purity is high (Figure 10 A).

B. be binding affinity and the specificity of the NIR dyestuff of target with folacin receptor. first use cancer (KB) cell of overexpression folacin receptor to evaluate affinity and the specificity of folic acid-and pteroic acid salt-NIR conjugate.Use tritium for the competitiveness research of folic acid (radiolabeled folic acid) show folic acid-or pteroic acid salt-NIR conjugate not only with high-affinity (low nanomole value) and also with high specific (Fig. 6) in conjunction with folacin receptor.Use tritium for the competitiveness research display Pte-Tyr-S0456 of folic acid (radiolabeled folic acid) with high-affinity and specific binding folacin receptor, this is enlightened large S0456 part and is puted together not containing infringement Pte-Tyr and folic acid (flote) receptors bind by phenol oxygen.

C. be the imaging of the NIR dyestuff of target with tumor in body. in order to more above-mentioned folic acid-NIR dye conjugate detects the ability of tumor, research and development xenograft models, this involves subcutaneous implantation KB cell (expressing the cell of FR), normally controls mice 4 weeks subsequently, grows to make nascent tumor.Then use the folic acid-NIR dye conjugate of the selection of 10nmol by the mice of tail vein injection treatment with tumor, after 2 hours, euthanasia is implemented, for fluorescence imaging to mice.As what observe in Fig. 7 A, be easy to distinguish knub position, produce between fluorescence cancer root nodule and adjacent healthy tissues and contrast by force.Most significantly, in the image of intact mice not opening or gather tumor, even also observe the tumor (Fig. 7 A) of complete fluorescence.The second filial generation is that head to head total body luminescence images research display folic acid-IR800CW (3) of the NIR reagent of target competes (in fluorescent brightness) (Fig. 7 A, second filial generation crude product) with other dyestuffs all with folacin receptor.But lamentedly, folic acid-IR800CW (3) not temperature in building-up process, causes the less desirable by-product formed more than 60%.As mentioned above, this will cause finding specific purification technique, its display path of higher production cost, the longer wait time limit for clinical conversion, and surgeon and patient cannot obtain this medicine.

In order to set up body internal specific, using Pte-Tyr-S0456 with on the mice shoulder of folate receptor-positive tumor xenogeneic graft.In body, whole body imaging research display Pte-Tyr-S0456 to be mainly accumulated in folate receptor-positive tumor and in other tissue, not to observe fluorescence (Figure 11 A).In vitro tissue biodistribution research display Pte-Tyr-S0456 is mainly accumulated in folate receptor-positive tumor, and essentially no fluorescence activity in other organ, except testis (Figure 11 B and 12B).Expection absorbs obviously in kidney, this is because the high-caliber FR of nearly tubule top film expression of known kidney.The head to head comparative study of Pte-Tyr-S0456 and folic acid-IR800CW, folic acid-LS288 and folic acid-ZW800 shows Pte-Tyr-S0456 and competes (Figure 12 A & B) with all folic acid-NIR conjugates in fluorescent brightness.Except, the fluoroscopic image enlightenment Pte-Tyr-S0456 of section (dissection) tumor and all folic acid-NIR conjugates are accumulated in equably and are even imbedded in (Figure 12 C) in all tumor cells of inside tumor.

The medicine in-vitro pharmacological research of embodiment 5 folic acid-and pteroyl--NIR dyestuff

Materials and methods:

KB cell (people's nasopharyngeal cells system) derives from American Type Culture preservation center (Rockville, MD) and use containing folic acid comprise 10% heat-inactivated fetal bovine serum (AtlantaBiological, GA) and 1% penicillin streptomycin (Gibco, NY) 1640RPMI culture medium (Gibco, NY) at 37 DEG C at 5% carbon dioxide: make it as monolayer growth at least 6 generation in 95% air wetting atmosphere, then they are used for measure.

The KB cell of overexpression FR-α is inoculated into 24-hole (100,000 cells/well) Falcon culture plate (BDBiosciences, CA), make it within the 12h time limit, form monolayer.By the culture medium that consumes in each hole and 10nM [ ³h]-folic acid (tritium is for folic acid) merging in fresh culture (0.5mL) under (0.1nM-1 μM) test article of progressive concentration or the existence of folic acid (Sigma-Aldrich, MO).At 37 DEG C after incubation 1h, rinse cell with PBS (3x0.5mL, Gibco, NY), to remove any unconjugated active material.In interpolation 0.25M sodium hydroxide (0.5mL) with after 4 DEG C of incubation 12h, cell is proceeded to and comprises Ecolite scintillation cocktail (3.0mL, MPBiomedicals, OH) each scintillation vial in, count with Liquid Scintillation Analyzer (Packard).Use the schematic diagram of the log concentration relationship of % cell bound radioactivity and test article, apply GraphPadPrism4 and calculate RA.

Result:

Dissociation constant (the K of this research will be derived from for compound OTL-001-OTL-0010 and folic acid _d) be calculated as 30.7nM, 19.3Nm, 23.3nM, 30.6nM, 50.1nM, 22.8nM, 30.5nM, 39.7nM, 49.6nM, 30.5nM and 8nM respectively.By RA, 0.270,0.430,0.356,0.271,0.166,0.364,0.272,0.209,0.167,0.272 and 1 is calculated as respectively for OTL-0001-OTL-0010 and folic acid.Whole test article quantitatively with [ ³h] competition of-folic acid.RA is defined as on alternative cell in conjunction with folacin receptor 50% [ ³h] mol ratio of compound needed for-folic acid; Relative affinity=1 of folic acid; Relative affinity <1 table four is more weak for the affinity of folacin receptor; Relative affinity >1 represent be combined with folacin receptor stronger.

Conclusion:

All compounds all have affinity for folacin receptor and they can appropriateness be drawn up with the binding affinity ratio of folic acid well.All compounds all with [ ³h]-folic acid competes fully, shows that folacin receptor constitutes binding site unique on cancerous cell and they have high degree of specificity for folacin receptor.

The medicine in-vitro pharmacological research of embodiment 6:OTL-0038 and OTL-0039 (the D-isomer of OTL-0038)

Research and develop two kinds of part-NIR conjugates and called after OTL-0038 and OTL-0039.OTL-0038 compound refers to PTE-L-Tyr-S0456, if pteroyl-is connected to S0456, then and part and TYR conjugate.OTL-0039 is the D-isomer of OTL-0038.Checked and to compare two kinds of compounds with the conjugated ligand folic acid of folacin receptor for the binding affinity of folacin receptor and binding specificity.

A. materials and methods

KB cell (people's nasopharyngeal cells system) derives from American Type Culture preservation center (Rockville, MD) and use containing folic acid comprise 10% heat-inactivated fetal bovine serum (AtlantaBiological, GA) and 1% penicillin streptomycin (Gibco, NY) 1640RPMI culture medium (Gibco, NY) at 37 DEG C at 5% carbon dioxide: make it as monolayer growth at least 6 generation in 95% air wetting atmosphere, then they are used for measure.

The KB cell of overexpression FR-α is inoculated into 24-hole (100,000 cells/well) Falcon culture plate (BDBiosciences, CA), make it in 12 hr period, form monolayer.By the culture medium that consumes in each hole and 10nM [ ³h]-folic acid (tritium is for folic acid) merging in fresh culture (0.5mL) under the OTL-0039 (D-isomer) of progressive concentration (0.1nM-1 μM) and the existence of OTL-0038 (L-isomer) or folic acid (Sigma-Aldrich, MO).At 37 DEG C, incubation is after 1 hour, rinses cell with PBS (3x0.5mL, Gibco, NY), to remove any unconjugated active material.In interpolation 0.25M sodium hydroxide (0.5mL) with at 4 DEG C of incubations after 12 hours, cell is proceeded to and comprises Ecolite scintillation cocktail (3.0mL, MPBiomedicals, OH) each scintillation vial in, count with Liquid Scintillation Analyzer (Packard).Use the schematic diagram of the log concentration relationship of % cell bound radioactivity and test article, apply GraphPadPrism4 and calculate RA.

B. result

Dissociation constant (the K of this research will be derived from for OTL-0039, OTL-0038 or folic acid _d) be calculated as 81.8nM, 10.4nM and 7.4nM respectively.By RA, 0.09,0.71 and 1 is calculated as respectively for OTL-0039, OTL-0038 and folic acid.Whole 3 kinds of test article quantitatively with [ ³h] competition of-folic acid.

RA is defined as on alternative cell in conjunction with folacin receptor 50% [ ³h] mol ratio of compound needed for-folic acid; Relative affinity=1 of folic acid; Relative affinity <1 table four is more weak for the affinity of folacin receptor; Relative affinity >1 represent be combined with folacin receptor stronger.

C. conclusion

OTL-0038 has affinity for folacin receptor and it can fully compare (10.4nM and 7.4nM) with the binding affinity of folic acid.On the other hand, when comparing with folic acid and OTL-0038, OTL-0039 has comparatively low-affinity for folacin receptor.OTL-0038 with [ ³h]-folic acid sufficient competition, show that folacin receptor forms the unique OTL-0038 binding site on cancerous cell and it has high degree of specificity to folacin receptor.

The whole body imaging of embodiment 7:OTL-0038 and OTL-0039 (the D-isomer of OTL-0038) in the mice with folacin receptor-positive tumor xenograft and bio distribution

Check that the folate receptor-positive tumor of OTL-0038 (PTE-L-Tyr-S0456) and OTL-0039 (PTE-D-Tyr-S0456) absorbs to determine that how two kinds of compounds are fully by the target acceptor absorbance in tumor.Also checked tissue biological's distribution of described compound.Intravenous uses half an hour after described compound, checks two kinds of characteristics of mice.

A. materials and methods

Cell culture and animal prepare

KB cell (people's nasopharyngeal cells system) derives from American Type Culture preservation center (Rockville, MD) and use containing folic acid comprise 10% heat-inactivated fetal bovine serum (AtlantaBiological, GA) and 1% penicillin streptomycin (Gibco, NY) 1640RPMI culture medium (Gibco, NY) at 37 DEG C at 5% carbon dioxide: make it as monolayer growth at least 6 generation in 95% air wetting atmosphere, then they are used for measure.

Athymic female naked (nu/nu) Mus (5 week age, 18-20g) purchased from HarlanLaboratories (Indianapolis, and maintained the folic acid-shortage specific meal (Teklad of gamma-irradiation IN), WI) on, at least 2 weeks, then start this research.Make animal reside in the bioclean of barrier with 5/cage to live away from home in framework.If need to give autoclaved tap water and food.Animal is made to reside in the time limit continuing this research in the gnotobasis of standard 12h fight-darkness cycle.Punched by ear and differentiate mice separately.Release animal care from sufferings and apply committee and have approved the operation of all animals.Animal care and research is carried out according to for the country of charitable animal process and international guidelines.

Whole body imaging

To on 7 week age female nu/nu mice shoulder through subcutaneous vaccination KB cell (in without the RPMI1640 culture medium of folic acid 1.0 × 10 ⁶/ mice).Used caliper measurements tumor growth in vertical direction (using same scheme to monitor body weight) every 2 days, gross tumor volume is calculated as 0.5 × L × W ²(the most major axis of L=, and the axle that W=and L is vertical, in millimeter).Once tumor reaches 400 to 500mm ³volume, then inject OTL-0038 or OTL-0039 in phosphate-buffered saline (100 μ L) of 10nmol to animal (5 mice/groups) through intravenous.After 2.5 hours, pass through CO ₂suffocate to euthanizing animals.Then the CaliperIVISLuminaIIImagingStation (PerkinElmerInc, MA) with LivingImage4.0 software is used to carry out whole body imaging (complete tumors) experiment.Imaging is arranged: the grade of-lamp: in; Excite: 745nm; Launch: ICG (indo cyanine green (indocyaninegreen)); Epi throws light on; Frame method: 4 (M), FOV=12.5; F-stops=2; Acquisition time=1s.

Tissue biological distributes

After whole body imaging, dissect animal, the fluorescence activity of the tissue (heart, lung, liver,spleen,kidney, stomach, small intestinal, large intestine, muscle, skin and tumor) using the analysis of IVIS imager to select as mentioned above.Imaging is arranged: the grade of-lamp: in; Excite: 745nm; Launch: ICG; Epi throws light on; Frame method: 4 (M), FOV=12.5; F-stops=2; Acquisition time=1s.

B. result

Whole body imaging

As viewed in Figure 14, OTL-0038 is mainly accumulated in folate receptor-positive tumor, essentially no fluorescence activity in other tissue.

Tissue biological distributes

Tissue biological's distributional analysis is carried out to the same animals by implementing euthanasia to every mice, taking out its organ and using the imaging of IVIS imager to carry out whole body imaging.As viewed in Figure 15, in FR-positive tumor, observe most high fluorescent, without accumulation in other tissue, except testis.Expection OTL-0038 absorbs in kidney, and this is owing to the high-caliber folacin receptor of top film expression of the proximal tubule of known kidney.Except, possible situation is, described probe passes through renal excretion because of its low-molecular-weight and half-life (most of folic acid conjugate has the half-life of <30min).

C. conclusion

OTL-0038 is mainly accumulated in folate receptor-positive tumor xenogeneic graft and kidney.Other normal structures all show floor level absorption or without absorption, cause the fluorescence of tumor and normal structure than splendid.

The relative analysis of the nearly IR reagent that embodiment 8:OTL-0038 (L-isomer) and folic acid derive

The whole body imaging of OTL-0038 and tissue biological's distribution are compared with folic acid-LS288, folic acid-IR800 and folic acid-ZW800.Make these compounds and folic acid and be purchased nir dye LS288, IR800 and ZW800 to put together.

A. materials and methods

Cell culture and mice prepare

KB cell (people's nasopharyngeal cells system) derives from American Type Culture preservation center (Rockville, MD) and use containing folic acid comprise 10% heat-inactivated fetal bovine serum (AtlantaBiological, GA) and 1% penicillin streptomycin (Gibco, NY) 1640RPMI culture medium (Gibco, NY) at 37 DEG C at 5% carbon dioxide: make it as monolayer growth at least 6 generation in 95% air wetting atmosphere, then they are used for measure.

Athymic female nude mice (5 week age, 18-20g) purchased from HarlanLaboratories (Indianapolis, IN) at least 2 weeks and in the folic acid-shortage specific meal (Teklad, WI) being maintained gamma-irradiation, then this research is started.Make animal reside in the bioclean of barrier with 5/cage to live away from home in framework.If need to give autoclaved tap water and food.Animal is made to reside in the time limit continuing this research in the gnotobasis of standard 12 h light-dark cycle.Punched by ear and differentiate mice separately.Release animal care from sufferings and apply committee and have approved the operation of all animals.Animal care and research is carried out according to for the country of charitable animal process and international guidelines.

Whole body imaging

To on 7 week age female nu/nu mice shoulder through subcutaneous vaccination KB cell (in without the RPMI1640 culture medium of folic acid 1.0 × 10 ⁶/ mice).Used caliper measurements tumor growth in vertical direction (using same scheme to monitor body weight) every 2 days, gross tumor volume is calculated as 0.5 × L × W ²(the most major axis of L=, and the axle that W=and L is vertical, in millimeter).Once tumor reaches 400 to 500mm ³volume, then inject the test article (OTL-0038, folic acid-LS288, folic acid-IR800, folic acid-ZW800) in phosphate-buffered saline (100 μ L) of 10nmol to animal (2 mice/groups) through intravenous.After 2.5h, pass through CO ₂suffocate to euthanizing animals.Then the CaliperIVISLuminaIIImagingStation (PerkinElmerInc, MA) with LivingImage4.0 software is used to carry out whole body imaging (complete tumors) experiment.Imaging is arranged: the grade of-lamp: in; Excite: 745nm; Launch: ICG (indo cyanine green); Epi throws light on; Frame method: 4 (M), FOV=12.5; F-stops=2; Acquisition time=1s.

Tissue biological distributes

After whole body imaging, dissect animal, the fluorescence activity of the tissue (heart, lung, liver,spleen,kidney, stomach, small intestinal, large intestine, muscle, skin and tumor) using the analysis of IVIS imager to select as mentioned above.Imaging is arranged: the grade of-lamp: in; Excite: 745nm; Launch: ICG; Epi throws light on; Frame method: 4 (M), FOV=12.5; F-stops=2; Acquisition time=1s.

B. result

Whole body imaging

As viewed in Figure 14, OTL-0038 (L-isomer), folic acid-LS288, folic acid-IR800, folic acid-ZW800 are mainly accumulated in folate receptor-positive tumor, essentially no fluorescence activity in other tissue.In addition, bright (higher) (Figure 15) of mice of the nearly IR dyestuff treatment of other folic acid of tumor fluorescence intensity ratio-put together of the mice of display injection OTL-0038 is directly compared.

Tissue biological distributes

Tissue biological's distributional analysis is carried out to the same animals by implementing euthanasia to every mice, taking out its organ and using the imaging of IVIS imager to carry out whole body imaging.As viewed in Figure 16, in FR-positive tumor and kidney, observe most high fluorescent.It is expection that kidney absorbs, and this is owing to the high-caliber folacin receptor of top film expression of the proximal tubule of known kidney.In addition, possible situation is, described probe passes through renal excretion because of its low-molecular-weight and half-life (most of folic acid conjugate has the half-life of <30min).

C. conclusion

Relative to folic acid-LS288, folic acid-IR800 and folic acid-ZW800, OTL-0038 thinks that the fluorescence intensity aspect that tumor is accumulated has beneficial aspects.It is bright that OTL-0038 is purchased nearly IR dyestuff such as LS288, IR800 and ZW800 than other.

The dose escalation study of embodiment 9:OTL-0038 in the mice with folate receptor-positive tumor xenogeneic graft

Carry out dosage ranging experiments to measure the lowest dose level can used and obtain best tumor and the OTL-0038 of background ratio.In addition, carry out testing to measure the maximum dose level can using to obtain best tumor (targeting) and the OTL-0038 of non-targeted tissue ratio.

A. materials and methods

KB cell (people's nasopharyngeal cells system) derives from American Type Culture preservation center (Rockville, MD) and use containing folic acid comprise 10% heat-inactivated fetal bovine serum (AtlantaBiological, GA) and 1% penicillin streptomycin (Gibco, NY) 1640RPMI culture medium (Gibco, NY) at 37 DEG C at 5% carbon dioxide: make it as monolayer growth at least 6 generation in 95% air wetting atmosphere, then they are used for measure.

Athymic female nude mice (nu/nu) (5 week age, 18-20g) purchased from HarlanLaboratories (Indianapolis, and maintained the folic acid-shortage specific meal (Teklad of gamma-irradiation IN), WI) on, at least 2 weeks, then start this research.Make animal reside in the bioclean of barrier with 5/cage to live away from home in framework.If need to give autoclaved tap water and food.Animal is made to reside in the time limit continuing this research in the gnotobasis of standard 12 h light-dark cycle.Punched by ear and differentiate mice separately.Release animal care from sufferings and apply committee and have approved the operation of all animals.Animal care and research is carried out according to for the country of charitable animal process and international guidelines.

To on 7 week age female nu/nu mice shoulder through subcutaneous vaccination KB cell (in without the RPMI1640 culture medium of folic acid 1.0 × 10 ⁶/ mice).Used caliper measurements tumor growth in vertical direction (using same scheme to monitor body weight) every 2 days, gross tumor volume is calculated as 0.5 × L × W ²(the most major axis of L=, and the axle that W=and L is vertical, in millimeter).Once tumor reaches 400 to 500mm ³volume, then inject the OTL-0038 (0.3nmol, 1nmol, 3nmol, 10nmol, 30nmol, 60nmol, 90nmol) in phosphate-buffered saline (100 μ L) of 10nmol to animal (3 mice/groups) through intravenous.After 2.5h, pass through CO ₂suffocate to euthanizing animals.Then the CaliperIVISLuminaIIImagingStation (PerkinElmerInc, MA) with LivingImage4.0 software is used to carry out tissue biological's distribution research.Imaging is arranged: the grade of-lamp: in; Excite: 745nm; Launch: ICG (indo cyanine green); Epi throws light on; Frame method: 4 (M), FOV=12.5; F-stops=2; Acquisition time=1s.

Injection 1nmolOTL-0038 after 2.5 hours, use the CaliperIVISLuminaIIImagingStation (PerkinElmerInc, MA) with LivingImage4.0 software to obtain whole body images (complete tumors).Imaging is arranged: the grade of-lamp: in; Excite: 745nm; Launch: ICG; Epi throws light on; Frame method: 4 (M), FOV=12.5; F-stops=2; Acquisition time=1s.

B. result

Shown in table 1 and Figure 14, all dosage all have in folate receptor-positive tumor higher tumor absorb, except 0.3nmol dosage.On the other hand, higher kidney is observed for 0.3-10nmol scope and absorbs, and less kidney absorption (absorbing relative to tumor) is observed to 30-90nmol scope.In addition, 60 and 90nmol observed at doses to higher non-specific adsorption.

C. conclusion

0.3nmol observed at doses to the lower absorption (hypofluorescence intensity) in folacin receptor-positive tumor can owing to not exclusively saturated on tumor cell of folacin receptor.On the other hand, the comparatively high fluorescent observed in kidney can be removed by kidney owing to described probe.In addition, the high-caliber folacin receptor of top film expression of the proximal tubule of known kidney.The dosage range display tumor of 1.0-30.0nmol absorbs and splendid tumor and normal structure ratio (signal and background compare).Higher kidney absorbs can remove (except 30nmol dosage) owing to described probe by kidney and folacin receptor is expressed on kidney.Dosage level 60.0nmol and lower than the higher non-specific adsorption of this value display.But.They still have high tumor and absorb and low kidney absorption (comprising 30nmol dosage).Lower kidney absorbs can be removed by liver and intestinal alternatively owing to described probe.Therefore, OTL-0038 can form aggregation with this higher concentration.We may safely draw the conclusion, 1.0nmol obtains good tumor with background than the lowest dose level used, it maintains the non-invasive aspect for tumor imaging (Figure 22) simultaneously, and 30nmol is for obtaining best tumor with background than the maximum dose level used.

The whole body imaging of embodiment 10:OTL-0038 in the mice with folacin receptor-negative tumours xenograft and bio distribution

Complete whole body imaging and tissue biological distribution to measure the body internal specific of OTL-0038 for folacin receptor.Experiment uses the hiding mice for the tumor of folacin receptor feminine gender to characterize the specificity of OTL-038 compound for folacin receptor.

A. materials and methods

Cell culture and mice prepare

A549 cell (alveolar Basal epithelial cancerous cell line) derives from American Type Culture preservation center (Rockville, MD) and use containing folic acid comprise 10% heat-inactivated fetal bovine serum (AtlantaBiological, GA) and 1% penicillin streptomycin (Gibco, NY) 1640RPMI culture medium (Gibco, NY) at 37 DEG C at 5% carbon dioxide: make it as monolayer growth at least 6 generation in 95% air wetting atmosphere, then they are used for measure.

Athymic female naked (nu/nu) Mus (6 week age, 18-20g) is purchased from HarlanLaboratories (Indianapolis, IN) and maintained in normal meals (Teklad, WI).Make animal reside in the bioclean of barrier with 5/cage to live away from home in framework.If need to give autoclaved tap water and food.Animal is made to reside in the time limit continuing this research in the gnotobasis of standard 12 h light-dark cycle.Punched by ear and differentiate mice separately.Release animal care from sufferings and apply committee and have approved the operation of all animals.Animal care and research is carried out according to for the country of charitable animal process and international guidelines.

Whole body imaging

To on 7 week age female nu/nu mice shoulder through subcutaneous vaccination A549 cell (in RPMI1640 culture medium 1.0 × 10 ⁶/ mice).Used caliper measurements tumor growth in vertical direction (using same scheme to monitor body weight) every 2 days, gross tumor volume is calculated as 0.5 × L × W ²(the most major axis of L=, and the axle that W=and L is vertical, in millimeter).Once tumor reaches 400 to 500mm ³volume, then inject the OTL-0038 in phosphate-buffered saline (100 μ L) of 10nmol to animal (6 mice/groups) through intravenous.After 2.5h, pass through CO ₂suffocate to euthanizing animals.Then the CaliperIVISLuminaIIImagingStation (PerkinElmerInc, MA) with LivingImage4.0 software is used to carry out whole body imaging (complete tumors) experiment.Imaging is arranged: the grade of-lamp: in; Excite: 745nm; Launch: ICG (indo cyanine green); Epi throws light on; Frame method: 4 (M), FOV=12.5; F-stops=2; Acquisition time=1s.

Tissue biological distributes

After whole body imaging, dissect animal, the fluorescence activity of the tissue (heart, lung, liver,spleen,kidney, stomach, small intestinal, large intestine, muscle, skin and tumor) using the analysis of IVIS imager to select as mentioned above.Imaging is arranged: the grade of-lamp: in; Excite: 745nm; Launch: ICG; Epi throws light on; Frame method: 4 (M), FOV=12.5; F-stops=2; Acquisition time=1s.

B. result

Whole body imaging

As viewed in Figure 14, OTL-0038 can not be accumulated in folacin receptor negative tumours, and essentially no fluorescence activity in other tissue, except testis.

Invasive tumor and kidney absorb

Tumor and kidney Accumulation Assay are carried out to the same animals by implementing euthanasia to every mice, taking out its organ and using the imaging of IVIS imager to carry out whole body imaging.Expect as us, in folacin receptor negative tumours, do not observe fluorescence, absorb at kidney camber.Since it is known the high-caliber FR of top film expression of the proximal tubule of kidney, so estimate that kidney absorbs.In addition, possible situation is, described probe passes through renal excretion because of its low-molecular-weight and half-life (most of folic acid conjugate has the half-life of <30min).

C. conclusion

OTL-0038 has high degree of specificity for folacin receptor.

Embodiment 11:OTL-0038 and OTL-0039 (the D-isomer of the OTL-0038) toxicity assessment in healthy nude mice

The toxicity in vivo of OTL-0038 and OTL-0039 is characterized in healthy mice.Often kind of compound of the clinical dosage of 1nmol or 1000x is used to check the toxicity of described compound to mice.

A. materials and methods

Athymic female naked (nu/nu) Mus (6 week age, 18-20g) is purchased from HarlanLaboratories (Indianapolis, IN) and maintained in normal meals (Teklad, WI).Make animal reside in the bioclean of barrier with 5/cage to live away from home in framework.If need to give autoclaved tap water and food.Animal is made to reside in the time limit continuing this research in the gnotobasis of standard 12 h light-dark cycle.Punched by ear and differentiate mice separately.Release animal care from sufferings and apply committee and have approved the operation of all animals.Animal care and research is carried out according to for the country of charitable animal process and international guidelines.

OTL-0038 or OTL-0039 being dissolved in 100 μ L phosphate-buffered saline being used 1 μm of ol fresh preparation Healthy female nude mice in 7 week age (5 mice/groups) by tail vein injection is given the 0th day time.Before administration, after this from the 0th day, body weight and clinical observation result was monitored to the 7th day every day.Any euthanizing animals of 20% or more can be lacked in continuous 2 days to body weight, but and nonessential.CO is passed through the 7th day time ₂suffocate to euthanizing animals, the tissue (heart, lung, liver,spleen,kidney, stomach, small intestinal, large intestine, muscle, skin) selected is gathered the bottle into comprising 4% formalin.The tissue fixed by formalin is cut into the section of 10 μm of thickness and is fixed on SuperfrostPlus ^tMon microscope slide (FisherScientific, PittsburghPA).After H & E stained slide, immunohistochemistry (IHC) is carried out to tissue and analyzes with the toxicity measuring OTL-0038 and OTL-0039.

B. result

After injection OTL-0038 or OTL-0039, animal skin becomes green at once.But, green disappearance in 24 hours.After using test article, animal quickens, normal to whole behavior to beginning in this research.As what observe in fig. 14, the stabilize body weight during this research.According to IHC data (Figure 15), in any tissue, all there is not the infringement of qualification.

C. conclusion

OTL-0038 or OTL-0039 of 1 μm of ol (1000x clinical dosage) is for animal avirulence, and this enlightenment OTL-0038 (1nmol) and OTL-0039 (1nmol) are clinically for people's avirulence.

Embodiment 12:

The medicine in-vitro pharmacological research of Pte-Tyrosine Analogues-S0456 (the OTL-0038 analog of modification)

Test article: OTL-0040 (Pte-Tyr- ¹³c-S0456), OTL-0042 (Pte-Tyr- ²h (deuterated)-S0456), OTL-0043 [Pte-Tyr-(OBn)-S0456], OTL-0044 [Pte-N (Me)-Tyr-S0456], OTL-0045 [Pte-NHNH-Tyr-(OAc)-S0456], OTL-0046 (high-Tyr-S0456 of Pte-), OTL-0047 (Pte-β-Gao-Tyr-S0456), OTL-0049 (Pte-Tyr amine-S0456)

Materials and methods: KB cell (people's nasopharyngeal cells system) derives from American Type Culture preservation center (Rockville, MD) and use containing folic acid comprise 10% heat-inactivated fetal bovine serum (AtlantaBiological, GA) and 1% penicillin streptomycin (Gibco, NY) 1640RPMI culture medium (Gibco, NY) at 37 DEG C at 5% carbon dioxide: make it as monolayer growth at least 6 generation in 95% air wetting atmosphere, then they are used for measure.

The KB cell of overexpression FR-α is inoculated into 24-hole (100,000 cells/well) Falcon culture plate (BDBiosciences, CA), make it in 12 hr period, form monolayer.By the culture medium that consumes in each hole and 10nM [ ³h]-folic acid (tritium is for folic acid) merging in fresh culture (0.5mL) under the test article of progressive concentration (0.1nM-1 μM) or the existence of folic acid (Sigma-Aldrich, MO).At 37 DEG C, incubation is after 1 hour, rinses cell with PBS (3x0.5mL, Gibco, NY), to remove any unconjugated active material.In interpolation 0.25M sodium hydroxide (0.5mL) with after 4 DEG C of incubation 12h, cell is proceeded to and comprises Ecolite scintillation cocktail (3.0mL, MPBiomedicals, OH) each scintillation vial in, count with Liquid Scintillation Analyzer (Packard).Use the schematic diagram of the log concentration relationship of % cell bound radioactivity and test article, apply GraphPadPrism4 and calculate RA.

Result:

Dissociation constant (the K of this research will be derived from for compound OTL-0040, OTL-0042-OTL-0047, OTL-0049 and folic acid _d) be calculated as 27.6nM, 61.7nM, 14.8nM, 13.8nM, 12.8nM, 30.2 and 8nM respectively.By RA, 0.290,0.130,0.177,0.580,0.625,0.265 and 1 is calculated as respectively for OTL-0040, OTL-0042-OTL-0047, OTL-0049 and folic acid.Whole test article quantitatively with [ ³h] competition of-folic acid.

Conclusion:

All compounds all have affinity to folacin receptor, except OTL-0044 and OTL-0045, and they can appropriateness compare with the binding affinity of folic acid well.All compounds all fully with [ ³h] competition of-folic acid, show that folacin receptor forms unique binding site on cancerous cell and they have high degree of specificity for folacin receptor.

Embodiment 13:

The whole body imaging of Pte-Tyrosine Analogues-S0456 and bio distribution

Test article: OTL-0043 [Pte-Tyr-(OBn)-S0456], OTL-0044 [Pte-N (Me)-Tyr-S0456], OTL-0045 [Pte-NHNH-Tyr-(OAc)-S0456], OTL-0046 (Pte-homo-Tyr-S0456), OTL-0047 (Pte-β-homo-Tyr-S0456), OTL-0049 (Pte-Tyr amine-S0456)

Materials and methods:

KB cell (people's nasopharyngeal cells system) derives from American Type Culture preservation center (Rockville, MD) and use containing folic acid comprise 10% heat-inactivated fetal bovine serum (AtlantaBiological, GA) and 1% penicillin streptomycin (Gibco, NY) 1640RPMI culture medium (Gibco, NY) at 37 DEG C at 5% carbon dioxide: make it as monolayer growth at least 6 generation in 95% air wetting atmosphere, then they are used for measure.

In athymic female nude mice (5 week age, 18-20g) at least 2 weeks purchased from Harlan (IN) and in the folic acid-shortage specific meal (Teklad, WI) being maintained gamma-irradiation, then start this research.Make animal reside in the bioclean of barrier with 5/cage to live away from home in framework.If need to give autoclaved tap water.Animal is made to reside in the time limit continuing this research in the gnotobasis of standard 12h fight-darkness cycle.Punched by ear and differentiate mice separately.Release animal care from sufferings and apply committee and have approved the operation of all animals.Animal care and research is carried out according to for the country of charitable animal process and international guidelines.

Whole body imaging:

To on 7 week age female nu/nu mice shoulder through subcutaneous vaccination KB cell (in without the RPMI1640 culture medium of folic acid 1.0 × 10 ⁶/ mice).Used caliper measurements tumor growth in vertical direction (using same scheme to monitor body weight) every 2 days, gross tumor volume is calculated as 0.5 × L × W ²(the most major axis of L=, and the axle that W=and L is vertical, in millimeter).Once tumor reaches 400 to 500mm ³volume, then inject the test article in phosphate-buffered saline (100 μ L) of 10nmol to animal (2-3 mice/group) through intravenous.After 2 hours, pass through CO ₂suffocate to euthanizing animals.Then the CaliperIVISLuminaIIImagingStation (PerkinElmerInc, MA) with LivingImage4.0 software is used to carry out whole body imaging (complete tumors) experiment.Imaging is arranged: the grade of-lamp: in; Excite: 745nm; Launch: ICG (830nm); Epi throws light on; Frame method: 4 (M), FOV=12.5; F-stops=2; Acquisition time=1s.

Tissue biological distributes:

After whole body imaging, dissect animal, the fluorescence activity of the tissue (heart, lung, liver,spleen,kidney, stomach, small intestinal, large intestine, muscle, skin and tumor) using the analysis of IVIS imager to select as mentioned above.Imaging is arranged: the grade of-lamp: in; Excite: 745nm; Launch: ICG (830nm); Epi throws light on; Frame method: 4 (M), FOV=12.5; F-stops=2; Acquisition time=1s.

Result:

Whole body imaging:

As viewed in Figure 28 A, OTL-0044, OTL-0046 and OTL-0047 are mainly accumulated in folacin receptor-positive tumor, essentially no fluorescence activity in other tissue.

Tissue biological distributes:

Under the same conditions to passing through to implement euthanasia to every mice, taking out its organ and use IVIS imager to carry out tissue biological's distributional analysis to its imaging.As viewed in Figure 28 B, in FR-positive tumor, observe most high fluorescent.It is expection that kidney absorbs, since it is known the high-caliber folacin receptor of top film expression of the proximal tubule of kidney.In addition, possible situation is, described probe passes through renal excretion because of its low-molecular-weight and half-life (most of folic acid conjugate has the half-life of <30min).

Conclusion:

Although bio distribution shows all compounds and is all accumulated in tumor and kidney in body, whole body distribution display OTL-0044, OTL-0046 and OTL-0047 are mainly accumulated in tumor, show the demand of α carboxylic acid for specificity and affinity.

The medicine in-vitro pharmacological research of embodiment 14:OTL-0050 (pteroyl--Tyr-S0122), OTL-0051 (pteroyl--Tyr-IRD28) and OTL-0052 (pteroyl--Tyr-Kodak)

Materials and methods:

KB cell (people's nasopharyngeal cells system) derives from American Type Culture preservation center (Rockville, MD) and use containing folic acid comprise 10% heat-inactivated fetal bovine serum (AtlantaBiological, GA) and 1% penicillin streptomycin (Gibco, NY) 1640RPMI culture medium (Gibco, NY) at 37 DEG C at 5% carbon dioxide: make it as monolayer growth at least 6 generation in 95% air wetting atmosphere, then they are used for measure.

The KB cell of overexpression FR-α is inoculated into 24-hole (100,000 cells/well) Falcon culture plate (BDBiosciences, CA), make it within the 12h time limit, form monolayer.By the culture medium that consumes in each hole and 10nM [ ³h]-folic acid (tritium is for folic acid) merging in fresh culture (0.5mL) under the test article of progressive concentration (0.1nM-1 μM) or the existence of folic acid (Sigma-Aldrich, MO).At 37 DEG C after incubation 1h, rinse cell with PBS (3x0.5mL, Gibco, NY), to remove any unconjugated active material.In interpolation 0.25M sodium hydroxide (0.5mL) with after 4 DEG C of incubation 12h, cell is proceeded to and comprises Ecolite scintillation cocktail (3.0mL, MPBiomedicals, OH) each scintillation vial in, count with Liquid Scintillation Analyzer (Packard).Use the schematic diagram of the log concentration relationship of % cell bound radioactivity and test article, apply GraphPadPrism4 and calculate RA.

Result:

Dissociation constant (the K of this research will be derived from for compound OTL-050-OTL-0052 and folic acid _d) be calculated as 29.3nM, 13.8nM, 15.3nM and 7.4nM respectively.By RA, 0.25,0.54,0.48 and 1 is calculated as respectively for OTL-0050-OTL-0052 folic acid.Whole test article quantitatively with [ ³h] competition of-folic acid.

Conclusion:

OTL-0050, OTL-0051 and OTL-0052 have affinity for folacin receptor separately and these compounds can appropriateness be compared with the binding affinity of folic acid well.All compounds all fully with [ ³h] competition of-folic acid, show that folacin receptor forms the unique binding site on cancerous cell and they have high degree of specificity for folacin receptor.

Embodiment 15: the medicine in-vitro pharmacological research of pteroyl--non-amino acid-NIR dye conjugate

Materials and methods:

KB cell (people's nasopharyngeal cells system) derives from American Type Culture preservation center (Rockville, MD) and use containing folic acid comprise 10% heat-inactivated fetal bovine serum (AtlantaBiological, GA) and 1% penicillin streptomycin (Gibco, NY) 1640RPMI culture medium (Gibco, NY) at 37 DEG C at 5% carbon dioxide: make it as monolayer growth at least 6 generation in 95% air wetting atmosphere, then they are used for measure.

The KB cell of overexpression FR-α is inoculated into 24-hole (100,000 cells/well) Falcon culture plate (BDBiosciences, CA), make it within the 12h time limit, form monolayer.By the culture medium that consumes in each hole and 10nM [ ³h]-folic acid (tritium is for folic acid) merging in fresh culture (0.5mL) under the test article of progressive concentration (0.1nM-1 μM) or the existence of folic acid (Sigma-Aldrich, MO).At 37 DEG C after incubation 1h, rinse cell with PBS (3x0.5mL, Gibco, NY), to remove any unconjugated active material.In interpolation 0.25M sodium hydroxide (0.5mL) with after 4 DEG C of incubation 12h, cell is proceeded to and comprises Ecolite scintillation cocktail (3.0mL, MPBiomedicals, OH) each scintillation vial in, count with Liquid Scintillation Analyzer (Packard).Use the schematic diagram of the log concentration relationship of % cell bound radioactivity and test article, apply GraphPadPrism4 and calculate RA.

Result:

Dissociation constant (the K of this research will be derived from for compound OTL-0056 (pteroyl--DAP-S0456), OTL-0057 (pteroyl--BAMB-S0456), OTL-0058 (pteroyl--AMHMB-S0456), OTL-0059 (pteroyl--DHDADS-S0456), OTL-0060 (pteroyl--DADS-S0456), OTL-0061 (pteroyl--4APEP-S0456) and folic acid _d) be calculated as 95.2nM, 121.3nM, 90.2nM, 250.5nM, 225.8nM, 41.7nM respectively.By RA, 0.078,0.061,0.082,0.029,0.033,0.171 and 1 is calculated as respectively for OTL-0056-OTL-0061 and folic acid.Whole test article quantitatively with [ ³h] competition of-folic acid.

Conclusion:

Compound OTL-0056-OTL-0061 has affinity for folacin receptor separately and they can appropriateness be compared with the binding affinity of folic acid well.All compounds all fully with [ ³h] competition of-folic acid, show that folacin receptor forms the unique binding site on cancerous cell and they have high degree of specificity for folacin receptor.

Embodiment 16:

The medicine in-vitro pharmacological research of pteroyl--non-amino acid-NIR dye conjugate

Materials and methods:

KB cell (people's nasopharyngeal cells system) derives from American Type Culture preservation center (Rockville, MD) and use containing folic acid comprise 10% heat-inactivated fetal bovine serum (AtlantaBiological, GA) and 1% penicillin streptomycin (Gibco, NY) 1640RPMI culture medium (Gibco, NY) at 37 DEG C at 5% carbon dioxide: make it as monolayer growth at least 6 generation in 95% air wetting atmosphere, then they are used for measure.

The KB cell of overexpression FR-α is inoculated into 24-hole (100,000 cells/well) Falcon culture plate (BDBiosciences, CA), make it within the 12h time limit, form monolayer.By the culture medium that consumes in each hole and 10nM [ ³h]-folic acid (tritium is for folic acid) merging in fresh culture (0.5mL) under the test article of progressive concentration (0.1nM-1 μM) or the existence of folic acid (Sigma-Aldrich, MO).At 37 DEG C after incubation 1h, rinse cell with PBS (3x0.5mL, Gibco, NY), to remove any unconjugated active material.In interpolation 0.25M sodium hydroxide (0.5mL) with after 4 DEG C of incubation 12h, cell is proceeded to and comprises Ecolite scintillation cocktail (3.0mL, MPBiomedicals, OH) each scintillation vial in, count with Liquid Scintillation Analyzer (Packard).Use the schematic diagram of the log concentration relationship of % cell bound radioactivity and test article, apply GraphPadPrism4 and calculate RA.

Result:

Dissociation constant (the K deriving from this research is calculated for compound OTL-0056-OTL-0061 and folic acid _d) and be determined as 95.2nM, 121.3nM, 90.2nM, 250.5nM, 225.8nM, 41.7nM and 7.4nM respectively.RA is calculated for OTL-0056 (Pte-DAP-S0456), OTL-0057 (Pte-BAMB-S0456), OTL-0058 (pteroyl--AMHMB-S0456), OTL-0059 (Pte-DHDADS-S0456), OTL-0060 (Pte-DADS-S0456), OTL-0061 (Pte-4APEP-S0456) and folic acid and is determined as 0.078,0.061,0.082,0.029,0.033,0.171 and 1 respectively.Whole test article quantitatively with [ ³h] competition of-folic acid.

Conclusion: all compounds all have weak affinity for folacin receptor.All compounds with [ ³h] competition of-folic acid, show that folacin receptor forms the unique binding site on cancerous cell and they have high degree of specificity for folacin receptor.

Embodiment 17:

The whole body imaging of pteroyl--aminoacid-NIR dye conjugate and bio distribution

Test article: OTL-0038 (Pte-Tyr-S0456), OTL-0053 (pteroyl--Lys-S0456) and OTL-0054 (pteroyl--Cys-S0456)

Materials and methods:

KB cell (people's nasopharyngeal cells system) derives from American Type Culture preservation center (Rockville, MD) and use containing folic acid comprise 10% heat-inactivated fetal bovine serum (AtlantaBiological, GA) and 1% penicillin streptomycin (Gibco, NY) 1640RPMI culture medium (Gibco, NY) at 37 DEG C at 5% carbon dioxide: make it as monolayer growth at least 6 generation in 95% air wetting atmosphere, then they are used for measure.

In athymic female nude mice (5 week age, 18-20g) at least 2 weeks purchased from Harlan (IN) and in the folic acid-shortage specific meal (Teklad, WI) being maintained gamma-irradiation, then start this research.Make animal reside in the bioclean of barrier with 5/cage to live away from home in framework.If need to give autoclaved tap water.Animal is made to reside in the time limit continuing this research in the gnotobasis of standard 12h fight-darkness cycle.Punched by ear and differentiate mice separately.Release animal care from sufferings and apply committee and have approved the operation of all animals.Animal care and research is carried out according to for the country of charitable animal process and international guidelines.

Whole body imaging:

To on 7 week age female nu/nu mice shoulder through subcutaneous vaccination KB cell (in without the RPMI1640 culture medium of folic acid 1.0 × 10 ⁶/ mice).Used caliper measurements tumor growth in vertical direction (using same scheme to monitor body weight) every 2 days, gross tumor volume is calculated as 0.5 × L × W ²(the most major axis of L=, and the axle that W=and L is vertical, in millimeter).Once tumor reaches 400 to 500mm ³volume, then inject the test article in phosphate-buffered saline (100 μ L) of 10nmol to animal (2-3 mice/group) through intravenous.After 2 hours, pass through CO ₂suffocate to euthanizing animals.Then the CaliperIVISLuminaIIImagingStation (PerkinElmerInc, MA) with LivingImage4.0 software is used to carry out whole body imaging (complete tumors) experiment.Imaging is arranged: the grade of-lamp: in; Excite: 745nm; Launch: ICG (830nm); Epi throws light on; Frame method: 4 (M), FOV=12.5; F-stops=2; Acquisition time=1s.When Pte-Lys-S0456, excite: 745nm, 710nm, 675nm, 640nm, 605nm; Launch: ICG, all the other parameters are identical.

Tissue biological distributes:

After whole body imaging, dissect animal, the fluorescence activity of the tissue (heart, lung, liver,spleen,kidney, stomach, small intestinal, large intestine, muscle, skin and tumor) using the analysis of IVIS imager to select as mentioned above.Imaging is arranged: the grade of-lamp: in; Excite: 745nm; Launch: ICG (830nm); Epi throws light on; Frame method: 4 (M), FOV=12.5; F-stops=2; Acquisition time=1s.

Result:

Whole body imaging:

As what observe in Figure 29, OTL-0038 (Pte-Tyr-S0456) and OTL-0054 (Pte-Cys-S0456) is mainly accumulated in folacin receptor-positive tumor, essentially no fluorescence activity in other tissue.In addition, the mice that other conjugate of tumor fluorescence intensity ratio directly comparing display OTL-0038 injection mice is treated is become clear.On the other hand, when exciting at 605-675nm wavelength and launch at ICG (830nm), Pte-Lys-S0456 has bright tumor fluorescence, and when exciting at 710-745nm and launch at ICG, only there is the brightness of appropriateness, as (the using identical parameter with other conjugate) observed in fig. 30.

Tissue biological distributes:

Under the same conditions to passing through to implement euthanasia to every mice, taking out its organ and use IVIS imager to carry out tissue biological's distributional analysis to its imaging.As viewed in Figure 31, in FR-positive tumor, observe most high fluorescent.It is expection that kidney absorbs, since it is known the high-caliber folacin receptor of top film expression of the proximal tubule of kidney.In addition, possible situation is, described probe passes through renal excretion because of its low-molecular-weight and half-life (most of folic acid conjugate has the half-life of <30min).

Conclusion:

From the best to the most weak conjugate listed the brightness of Ex=745nm and Em=ICG and specificity as follows: Pte-Tyr-S0456, Pte-Cys-S0456 and Pte-Lys-S0456.Pte-Lys-S0456 all shows longer Stoke ' sshift, shows that we can excite at 605nm and launch to observe bright tumor fluorescence at ICG (830nm).

Embodiment 18:

The whole body imaging of Pte-Tyr-Kodak derivant [OTL-0051 (pteroyl--Tyr-IRD28) and OTL-0052 (pteroyl--Tyr-Kodak)] and bio distribution

Test article: OTL-0051 (pteroyl--Tyr-IRD28) and OTL-0052 (pteroyl--Tyr-Kodak)

Materials and methods:

KB cell (people's nasopharyngeal cells system) derives from American Type Culture preservation center (Rockville, MD) and use containing folic acid comprise 10% heat-inactivated fetal bovine serum (AtlantaBiological, GA) and 1% penicillin streptomycin (Gibco, NY) 1640RPMI culture medium (Gibco, NY) at 37 DEG C at 5% carbon dioxide: make it as monolayer growth at least 6 generation in 95% air wetting atmosphere, then they are used for measure.

In athymic female nude mice (5 week age, 18-20g) at least 2 weeks purchased from Harlan (IN) and in the folic acid-shortage specific meal (Teklad, WI) being maintained gamma-irradiation, then start this research.Make animal reside in the bioclean of barrier with 5/cage to live away from home in framework.If need to give autoclaved tap water.Animal is made to reside in the time limit continuing this research in the gnotobasis of standard 12h fight-darkness cycle.Punched by ear and differentiate mice separately.Release animal care from sufferings and apply committee and have approved the operation of all animals.Animal care and research is carried out according to for the country of charitable animal process and international guidelines.

Whole body imaging:

To on 7 week age female nu/nu mice shoulder through subcutaneous vaccination KB cell (in without the RPMI1640 culture medium of folic acid 1.0 × 10 ⁶/ mice).Used caliper measurements tumor growth in vertical direction (using same scheme to monitor body weight) every 2 days, gross tumor volume is calculated as 0.5 × L × W ²(the most major axis of L=, and the axle that W=and L is vertical, in millimeter).Once tumor reaches 400 to 500mm ³volume, then inject the test article in phosphate-buffered saline (100 μ L) of 10nmol to animal (2-3 mice/group) through intravenous.After 2 hours, pass through CO ₂suffocate to euthanizing animals.Then the CaliperIVISLuminaIIImagingStation (PerkinElmerInc, MA) with LivingImage4.0 software is used to carry out whole body imaging (complete tumors) experiment.Imaging is arranged: the grade of-lamp: in; Excite: 745nm; Launch: ICG (830nm); Epi throws light on; Frame method: 4 (M), FOV=12.5; F-stops=2; Acquisition time=1s.When Pte-Lys-S0456, excite: 745nm, 710nm, 675nm, 640nm, 605nm; Launch: ICG (830nm), all the other parameters are identical.

Tissue biological distributes:

After whole body imaging, dissect animal, the fluorescence activity of the tissue (heart, lung, liver,spleen,kidney, stomach, small intestinal, large intestine, muscle, skin and tumor) using the analysis of IVIS imager to select as mentioned above.Imaging is arranged: the grade of-lamp: in; Excite: 745nm; Launch: ICG (830nm); Epi throws light on; Frame method: 4 (M), FOV=12.5; F-stops=2; Acquisition time=1s.

Result:

Whole body imaging:

As what observe in Figure 32, OTL-0051 (pteroyl--Tyr-IRD28) and OTL-0052 (pteroyl--Tyr-Kodak) is mainly accumulated in folacin receptor-positive tumor, essentially no fluorescence activity in other tissue.Although Kodak dyestuff excites at 800nM, IVIS image system does not have the filter excited at 800nM.Charming from, the low Poison observed in tumor absorbs can owing to applying weak excitation wavelength.

Tissue biological distributes:

Under the same conditions to passing through to implement euthanasia to every mice, taking out its organ and use IVIS imager to carry out tissue biological's distributional analysis to its imaging.As viewed in fig. 33, in FR-positive tumor, observe most high fluorescent.We also observe the absorption in lung.Although our expection exists kidney absorb (since it is known high-caliber folacin receptor of top film expression of the proximal tubule of kidney), kidney degree of absorption is low.

Conclusion:

Tissue biological's distribution research display OTL-0051 (pteroyl--Tyr-IRD28) and OTL-0052 (pteroyl--Tyr-Kodak) conjugate absorb in folate receptor-positive tumor.

Claims (69)

1. there is the compound of following formula:

Or its pharmaceutically acceptable salt,

Or its isotope,

Wherein:

X is aminoacid or derivatives thereof, and

Y is the dyestuff near infrared range with fluorescence excitation and emission spectra, and this compound maintains or strengthens the fluorescence of described dyestuff.

2. the compound of claim 1, wherein said aminoacid is selected from the derivant of tyrosine, cysteine or tyrosine or cysteine.

3. the compound of claim 1, wherein said aminoacid is tyrosine.

4. the compound of claim 3, wherein the isotope of carbon is positioned on the aromatic ring of tyrosine.

5. the compound of claim 4, wherein the isotope of hydrogen is the substituent group of the aromatic ring of tyrosine.

6. the compound of claim 1, wherein said amino acid derivativges is tyrosine derivative, and it is selected from:

Or its racemic mixture.

7. the compound of claim 1, wherein Y has following formula:

Wherein

X independently selected from O, S, N and C, and

R is independently selected from CH ₂and CH ₂cH ₂.

8. the compound of claim 1, wherein said aminoacid comprises sulfur-containing side chain base.

9. the compound of claim 8, the aminoacid comprising sulfur-containing side chain base is cysteine.

10. the compound of claim 8, the aminoacid comprising sulfur-containing side chain base is methionine.

The compound of 11. claim 1 is selenocysteine comprising the aminoacid containing chalcogen side chain radical.

The compound of 12. claim 1, wherein this compound has following formula:

Or its potassium, sodium or ammonium salt.

The compound of 13. claim 1, wherein this compound has following formula:

Or its pharmaceutically acceptable salt.

The compound of 14. claim 1, wherein this compound has following formula:

Or its pharmaceutically acceptable salt.

The compound of 15. claim 1, wherein this compound has absorption maximum and the transmitting of about 500nm to about 900nm.

The compound of 16. claim 15, wherein this compound has absorption maximum and the transmitting of about 600nm to 800nm.

The compound of 17. claim 1, sends fluorescence after wherein making this compound distribute in histiocyte.

The compound of 18. claim 1, wherein sends fluorescence by making this compound contact the exciting light of near-infrared wavelength.

The compound of 19. claim 1, wherein this compound has the binding affinity to folacin receptor target, similar with the binding affinity of the folic acid to folacin receptor.

The compound of 20. claim 1, wherein this compound has the near-infrared brightness of the folic acid conjugate being greater than compound, and described compound is selected from:

The compound of 21. claim 1, wherein said compound high selectivity ground targets neoplastic cells.

22. compositionss, comprise compound and pharmaceutically acceptable carrier, excipient or the diluent of claim 1.

23. methods making the biological organism optical imaging of expression folacin receptor, the method comprises:

A () makes the compositions of described contact biological tissue claim 22;

B time that () allows the compound in compositions to distribute in biological target;

C () organizes described in the excitation light irradiation of the absorbable wavelength of this compound; With

D () detects the optical signalling that this compound is launched.

The method of 24. claim 23, the signal that wherein said compound is launched is used for design of graphics picture.

The method of 25. claim 23, is wherein saidly organized in experimenter, and described experimenter is animal or human.

The method of 26. claim 23, wherein in step (a), makes its characteristics of signals two or more fluorescent chemicalses differentiable contact described tissue, is organized in experimenter optionally.

The method of 27. claim 25, wherein irradiates and detecting step uses microscope in endoscope, conduit, x-ray tomography system, portable optical imaging system, surgical operation protective eye lens or operation to carry out.

The method of 28. claim 23, the biological tissue of wherein expressing folacin receptor has the disease of overexpression this receptor.

The method of 29. claim 28, wherein said disease selected from cancer, cardiovascular disease, neurodegenerative disease, immunological diseases, autoimmune disease, respiratory system disease, metabolic disease, genetic diseases, infectious disease, osteopathia and environmental disease.

The method of target cell type in 30. identification of organism samples, comprising:

A) make described biological sample contact the compound of claim 1, time of contact and this compound of conditions permit are in conjunction with at least one cell of target cell type; With

B) compound described in presence or absence in described biological sample is optionally detected,

Wherein at detecting step b) in there is described compound and represent that described target cell type is present in described biological sample.

The method of 31. claim 30, wherein said target cell type is the tissue for diagnostic purpose to be imaged.

The method of 32. claim 31, wherein said tissue is tumor or lymph node.

The method of 33. claim 30, wherein said optionally detection comprises to be made described compound contact excitation source and detects the fluorescence from this compound.

The method of 34. claim 33, wherein said exciting light is the light of near-infrared wavelength.

The method of 35. claim 33, wherein said excitation wavelength is in about 600 to 1000 nanometer range.

The method of 36. claim 33, wherein said excitation wavelength is in about 670 to 850 nanometer range.

37. couples of experimenters implement the method for the operation that image instructs, and comprising:

A) use the compositions of the compound comprising claim 1, application conditions and time are enough to make described compound be accumulated on given surgical position;

B) described compound is irradiated to use infrared light to make this compound develop; With

C) excision is implemented for by the region sending fluorescence time infrared ray excited.

The method of 38. claim 37, its mid-infrared light is the light of near-infrared wavelength.

The method of 39. claim 37, the wavelength of its mid-infrared light is in about 600 to 1000 nanometer range.

The method of 40. claim 37, the wavelength of its mid-infrared light is in about 670 to 850 nanometer range.

The method of the disease of 41. diagnosis experimenters, comprising:

A) to needing the experimenter of diagnosis to use the compound of a certain amount of claim 1, time of application and this compound of conditions permit are in conjunction with at least one cell of target cell type;

B) signal from the compound of the claim 1 be present in biological sample is measured;

C) compared with at least one contrasting data group by the signal measured in b), at least one contrasting data group wherein said comprises the signal of the compound from claim 1, and this compound contacts the biological sample not containing target cell type; With

D) provide the diagnosis of disease, wherein step c) in comparison indicate the existence of described disease.

42. test kits, comprise the compound of claim 1.

The test kit of 43. claim 42, wherein the compound of claim 1 produces fluorescence signal, and the brightness of this fluorescence signal is greater than the brightness of the fluorescence signal of the compound of the folic acid conjugate of inclusion compound, and described compound is selected from:

The test kit of 44. claim 43, also comprises the derivant of the compound of claim 1.

The compound of 45. claim 44, wherein Y is the compound with following formula:

Wherein Rx is selected from sulfate, sulfonate, phosphate, phosphonate, ammonium and hydroxyl, sugar, aminoacid, sulfonamide or carboxyl independently of one another.

The compound of 46. claim 43, wherein Y has following formula:

Wherein

X independently selected from O, S, N or C, and

R is independently selected from CH ₂or CH ₂cH ₂.

The compound of 47. claim 1, wherein this compound has and is selected from following formula:

Wherein W, X, Y or Z are H, Na or NH ₄ ⁺,

Wherein W, X, Y or Z are H, Na or NH ₄ ⁺,

Wherein W, X, Y or Z are H, Na or NH ₄ ⁺,

Wherein W, X, Y or Z are H, Na or NH ₄ ⁺,

Wherein W, X, Y or Z are H, Na or NH ₄ ⁺,

Wherein tyrosine is β height tyrosine,

Wherein W, X, Y or Z are H, Na or NH ₄ ⁺,

And pharmaceutically acceptable salt.

48. compounds, have following formula:

Or its pharmaceutically acceptable salt,

Or its isotope,

Wherein:

X is aminoacid or derivatives thereof, and

Y is the dyestuff with fluorescence excitation in the visible spectrum and the emission spectra near infrared range, and X is connected to Y, generates the secondary amine being connected directly to Y, and described compound maintains or strengthens the fluorescence of described dyestuff.

The compound of 49. claim 48, wherein said aminoacid is naturally occurring aminoacid.

The compound of 50. claim 48, wherein said aminoacid is alpha amino acid.

The compound of 51. claim 50, wherein said aminoacid is homoamino acid.

The compound of 52. claim 48, wherein said aminoacid is beta amino acids.

The compound of 53. claim 48, wherein said aminoacid comprises the side chain radical of hydroxyl.

The compound of 54. claim 53, wherein said aminoacid is selected from serine, tyrosine, threonine, its isomer and derivant thereof.

The compound of 55. claim 48, wherein said aminoacid comprises containing oxygen side chain radical.

The compound of 56. claim 55, wherein said aminoacid is selected from glutamine, glutamic acid, glutamate, Glu, agedoite, its isomer and derivant thereof.

The compound of 57. claim 48, wherein said aminoacid comprises aromatic side chains base.

The compound of 58. claim 57, the aminoacid comprising aromatic side chains base is selected from phenylalanine, tryptophan and tyrosine.

The compound of 59. claim 48, wherein said aminoacid comprises sulfur-containing side chain base.

The compound of 60. claim 59, the aminoacid comprising sulfur-containing side chain base is cysteine.

The compound of 61. claim 48 is selenocysteine comprising the aminoacid containing chalcogen side chain radical.

The compound of 62. claim 48, wherein said aminoacid is neutral polar amino acid.

The compound of 63. claim 62, wherein said aminoacid is selected from glycine, serine, threonine, cysteine, tyrosine, agedoite and glutamine.

The compound of 64. claim 48, wherein said aminoacid comprises the side chain radical containing amine.

The compound of 65. claim 64, wherein said amino acid selected from lysine, arginine, agedoite, glutamine, histidine and isomer thereof and derivant.

The compound of 66. claim 48, wherein said amino acid derivativges comprises basic side chain base.

The compound of 67. claim 66, wherein said amino acid selected from lysine, arginine and histidine.

The compound of 68. claim 48, wherein said amino acid derivativges comprises containing chalcogen side chain radical.

The compound of 69. claim 68, wherein chalcogen is selected from oxygen, sulfur and selenium.